biomedical implications of hormesis – part...

A Publication of the Northeast Regional Environmental Public Health Center, University of Massachusetts, School of Public Health, Amherst, MA 01003Vol. 13 No. 2 Part I, September 2005, ISSN 1092-4736

Biological Effects ofLow Level Exposures

BIOMEDICAL IMPLICATIONS OFHORMESIS – PART 1

TABLE OF CONTENTS

INTRODUCTION: BIOMEDICAL IMPLICATIONS OFHORMESIS – PART1 Edward Calabrese...............................1

SHOULD WE EXPLORE THE CLINICAL UTILITY OFHORMESIS? Wayne B. Jonas................................................2

THE MANAGED IMMUNE SYSTEM: PROTECTING THEWOMB TO DELAY THE TOMB Rodney R. Dietert andMichael S. Piepenbrink..........................................................7

LOW-DOSE RADIATION AND ITS CLINICAL IMPLICA-TIONS: DIABETES Guan-Jun Wang, Xiao-Kun Li,Kazuo Sakai and Lu Cai.....................................................12

BREAST CANCER, CHEMOTHERAPY AND HORMESISLorne J. Brandes.................................................................22

NONLINEARITY IN BIOLOGY, TOXICOLOGY ANDMEDICINE JOURNAL.....................................................28

INTERNATIONAL HORMESIS SOCIETY .......................28

IHS APPLICATION FOR MEMBERSHIP..........................29

ADVISORY COMMITTEE................................................31

While the hormesis concept has often beenassessed within the context of its riskassessment implications, it is becomingmore widely recognized that will also haveat least as large and significant animpact on the biomedical sciences. Con-sequently, this issue of the BELLE News-letter is devoted to an exploration of someof the biomedical implications of hormesis.Since we believe that these articles repre-sent only the so-called tip of the iceberg interms of biomedical implications ofhormesis, we hope that this issue willencourage others to explore their areas ofinvestigation. It is our hope that im-proved knowledge of the dose response andtheir interactions with adaptive mecha-nisms will enable researchers to exploreand find practical ways to enhance thehealth of patients and entire populations.The present contributions not only providedetailed and integrative information onthis topic but also often point the directionwhere important developments are likely.

INTRODUCTION

2 BELLE Newsletter

SHOULD WEEXPLORE THECLINICAL UTILITYOF HORMESIS?Wayne B. Jonas, MD

Director, Samueli Institute for Information Biology

1700 Diagonal Road

Suite 400

Alexandria, Virginia 22314

Phone: 703-299-4800

Fax: 703-535-6752

Email: [email protected]

John A. Ives, PhD

Samueli Institute for Information Biology

1700 Diagonal Road

Suite 400

Alexandria, Virginia 22314

Phone: 703-299-4800

Fax: 703-535-6752


ABSTRACT

The idea that low-dose adaptive effects as described inhormesis can be used clinically has been discussed forhundreds if not thousands of years. Paracelsus famousadage that “the dose makes the poison” and the commonfolk saying that one can be cured by “the hair of the dogthat bit you” speak to this idea. So why has so littleresearch been done on the possible clinical utility ofhormesis? What areas of clinical hormesis seem to be themost promising to explore? This article examines theseconcepts and proposes some initial areas of researchwhere the possible utility of hormeiss might be investi-gated.

WHY HAS CLINICAL HORMESIS NOTBEEN DEVELOPED?Given the growing literature on hormesis and its poten-tially wide clinical utility, why has research on the clinicaleffects of hormetic dose-responses not been moreextensively explored? We propose that a number ofobstacles to the investigation of clinical hormesis haveprevented its development. These include an almostexclusive use of the term in toxicology, the inaccuratedefinition of hormesis in its early years, the association of

hormesis with the fringe medical system called homeopa-thy, the powerful effects of pharmacology at high doses,and the disastrous effects of early, unscientific attemptsto clinically apply radiation hormesis.

Use and Definitions of Hormesis in Toxicology

The term hormesis was coined by two toxicologists in1943 and its discussion has, until recently, remained as asmall discussion in the toxicology literature. 1 Its implica-tions remain focused mostly around environmental riskassessment. Only recently has the term been used in astandard textbook of pharmacology, yet most pharma-cologists we speak to are not familiar with the term. Theinitial definitions of hormesis revolved around stimula-tory effects of low-doses of toxins often emphasizing anapparent positive rather than adaptive, nature of sub-stances that were normally associated with adverseeffects. 2 This was interpreted by some skeptics as anattempt to push the beneficial nature of the area ratherthan more precisely define and understand the science.When Ed Calabrese and Linda Baldwin, two toxicolo-gists, began to show that hormetic dose-responses werewidespread outside the field of toxiciology the discussionbegan to take on a more interdisciplinary nature, withthe recent creation of the professional association theInternational Hormesis Society. 3 Still, few physicians orclinical investigators are involved in this Society or thefield.

Association with Homeopathy

In contrast to toxicologists’ failure to discuss the clinicalimplications of hormesis, practitioners of the fringemedical system called homeopathy were more thanwilling to use the concept in an attempt to justify theirpractice. Schulz, one of the first to describe thehormetic phenomenon believed he had discovered theunderlying mechanism of homeopathy. This furthercontaminated any discussion of possible clinical use ofthe concept. 2 Homeopaths were ousted from main-stream medicine and often fought with conventionalpractitioners. In addition, they held to the irrationalclaim that dilutions beyond Avogadro’s number still hadeffects. 4 Some of their drugs were eventually founduseful in conventional medicine, although at higherdoses. Instead of clearly differentiating the study ofclinical hormesis from homeopathy, however, many inthe field of hormesis avoided the concept all together,refusing to discuss or explore any clinical applications.This is unfortunate since the rising use of complemen-tary and alternative medicine has created an increasedinterest in the low-dose effects of chemicals in foods,herbs, and homeopathic substances as well as otherstimulatory interventions such as exercise and psycho-therapy. 5, 6 Hormesis may offer insights into the mecha-nisms of some of these therapies. For example, it ispossible that the reported effects from ultra-high dilu-tions in homeopathy are due to hormetic dose-responses

Vol. 13, No. 2 Part I, September 2005 3

secondary to the borosilicate contaminants derived fromthe glass in which these preparations are made. Mosthomeopathic medicines are made in glass and significantamounts of borosilicates contaminate their solutions. 7

Silicates are often bioactive. Thus, a likely explanationfor the reported effects in homeopathy is secondarysilicate contamination.

The Power of Pharmacology

It was around the same time the phenomenon ofhormesis was being explored in toxicology, that thepowerful effects of high dose pharmacology was beingdemonstrated and widely used. These discoveries over-shadowed any interest in the clinical effects of low doses.Antibiotics, anesthetics, analgesics and chemotherapeu-tic agents produced such dramatic effects that the almostexclusive focus in pharmacology was on the discovery ofnew therapeutic agents and agents with lower toxicity,not the potential use of low-doses of drugs. At the sametime, misguided attempts to apply radiation hormesisresulted in disaster. When Eben Byers, a millionairepromoter of a radioactive “longevity tonic” calledRadithor, died of radiation poisoning in 1932, the idea ofclinical applications of hormesis took another hit, sincehe had promoted the tonic as scientifically provenbecause of hormesis. 8

RESEARCH ON THE POSSIBLECLINICAL UTILITY OF HORMESIS

There is a growing literature on exploring the possibleutility of hormetic dose-response effects. Ed Calabreseand Linda Baldwin have summarized several areas ofhormesis that may have clinical implications and applica-tions. Alzheimer’s, bone remineralization, tumor growthand revascularization, hair growth, and viral infectionsall have evidence of hormesis as an approach to treat-ment. 9 A recent special issue in Critical Reviews in Toxicol-ogy focused on hormetic dose-response relationships inimmunology including its use in bacterial and viraldisease, lupus, Grave’s disease, acute respiratory diseases,and cancer. 10 Hormetic dose-response relationships maybe the basis for treatment outside toxicology in areas ofpsychology and stress management. 11

There may be wide potential for clinical applications ofhormesis. It is now well established that low-doses oftoxic and infectious agents often stimulate growth, repairand protective processes in cells, animals and humans.These effects are found to occur with a number oftoxins, drugs, viral, and bacterial agents includingenvironmental toxins and those used for chemical,biological, nuclear and radiation [CBRN] terroristpurposes. 12 Hormetic responses occur in various celllines, tissues, animal and plant species and humans andhave been observed after exposure to low levels of toxicchemicals 3, 13, including heavy metals 14, infectious agents

15, as well as ionizing radiation 16 and in trauma. 17, 18

Exposure to low-level stressors can induce both generaland specific protective effects. Studies have demon-strated this occurs from exposure to ischemia, heat,hydrogen peroxide, nicotine, oxygen radicals, alcohol,heavy metals (e.g., cadmium, arsenic, lead), cytotoxicand carcinogenic agents used for chemotherapy (e.g.,adriamycin, cisplatinum), interleukin-1 (and othercytokines), gram-negative organisms and other stressors.3, 18, 19 Reduced mortality as well as reduced cellular andorgan damage has been found in brain, liver, kidney,lung, muscle, and in a number of isolated cell lines. Is itpossible to use such low-dose exposures to rapidly induceprotective and therapeutic effects to toxic exposures andstresses without causing harm? 20 Protective effects canoccur well below the no observable adverse effect level(NOAEL). Could hormesis offer an alternative approachfor the mitigation of a number of toxic and infectiousagents but with potentially wider application, safety andflexibility than current vaccine and anti-toxin ap-proaches?

There are a number of examples whereby exposure tosub-toxic doses of otherwise toxic compounds confersprotection and treatment against higher toxic doses ofthe same or similar harmful compounds. 20 We call thisconcept Rapid Induction of Protective Tolerance(RIPT). RIPT occurs by inducing a stimulatory effect oncell repair, tolerance and protective processes. Onechallenge in the study of this area is that significantclinical effects would likely arise from a coordinatedwhole organism response of inherent [self-derived]healing and defense processes that are complex toinvestigate. The clinical value of hormesis may be mostevident if multiple, redundant mechanisms are induced.21, 9 Many cellular protective mechanisms are distinct fromimmune stimulation, like that produced by vaccines, yetimmune mechanisms may enhance and extend a RIPTeffect. 10 If true, this would allow rapid use of hormesis ina wide variety of situations including terrorism, environ-mental toxicity, drug toxicity, cancer and emerginginfections such as influenza, SARS and Avian flu.

RECENT RESEARCH ON POTENTIALAPPLICATIONS OF HORMESIS

We have been investigating the potential protectiveeffects of low level exposure using a number of toxins ina variety of studies. Our program on protective andtreatment effects examines the biological effects of lowlevel exposures to physical, chemical, and biologicalagents as a simple method for modulating the adverseeffects from exposure to higher doses of the same toxins.22 The method could be applicable to self-treatment oftoxin exposure by soldiers on the battlefield and forprotecting civilian populations from terrorist

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attack as well as in other biomedical areas. Potentialareas of application in need of further investigationinclude the following.

Terrorism and Biowarfare Protection

There is evidence that rapid induction of protectivetolerance (RIPT) against biowarfare and terrorists agentsmay be feasible. 23 An early double-blind clinical study ofthe biowarfare agent mustard gas demonstrated that low-dose mustard gas and similar blistering agents reduceddamage to mustard in humans. 24 Little research on theconcept followed, however. We recently did a compre-hensive, systematic review of the CBRN literature forstudies examining the stimulatory and protective effectsfor the top 10 CBRN terrorist agents. 25 The area is rarelyinvestigated, but most studies that specifically looked forstimulatory or protective effects, found them, includingwith the potent neurotoxins soman and sarin. Jonas andDillner 15 investigated whether low-dose preparations ofinfected tissue given to mice could induce protectionagainst a higher infectious challenge by the same organ-ism. Their data demonstrated that these preparationsconsistently increase mean survival time and decreasethe mortality from Francisella tualrensis infection, a topbiowarfare threat agent.

Brain Injury

Recently, Jonas, Tortella and Ives 17, 18, 26, 27 have shown thatRIPT can be used to reduce brain injury after trauma.Low dose glutamate [the primary toxin released duringbrain injury] reduces core brain damage by 40% inanimal brain injury models. By complexing glutamatewith low-dose silica particles its effectiveness was mark-edly enhanced through induction of several classes ofproteins. 18 28 Importantly, brain damage was reducedeven when low dose glutamate/silica was administeredafter the trauma providing evidence that the RIPTconcept may be a useful approach for both preventionand treatment in brain injury and stroke. The combina-tion of a low-dose toxin with silica appears to be a keymechanism for enhancement of the effects of varioustoxic agents when given at low-dose and through thestimulation of protective proteins. As mentioned above,silica-toxin complexes may also provide a rationalexplanation for the reported effects of homeopathyrather than unplausible explanations such as “memoryin water.”

Environmental Toxins and Cancer

The RIPT approach may also help mitigate the effects ofenvironmental toxins such as arsenic, mercury andcadmium. We conducted a meta-analysis of the literatureon the low-dose protective effects of environmentaltoxins of various types. 29, 30 Significant protective effectswere demonstrated in repeated studies with arsenic andmercury, two of the most important environmental

toxins worldwide. Low dose arsenic and mercury en-hanced toxin excretion up to 40% and reduced mortalityto lethal doses by 19%. These findings have recentlybeen confirmed by others 31. Similar work with cadmiumis reported in several studies. 32, 33 As with CBRN andbrain injury agents, the rapid induction of protectiveproteins appears to be an important mechanism. VanWijk demonstrated that specific patterns of heat shockproteins [hsp] predicted cross protection to a variety ofenvironmental toxins. 22 We have shown in our laborato-ries that non-toxic, low-dose cadmium exposure rapidlystimulates specific methallothienien production [aprotective protein] and its mRNA signal, which can bemaintained for weeks 32 with no adverse effects on cellgrowth, replication, function or mortality. Subsequentexposure of the same cells to higher doses of cadmiumshowed delayed transformation into cancer usuallyproduced by cadmium. Thus, a “window of protection”to specific agents can be turned on and off for weeks at atime apparently without harm.

Emerging Infections

Many infectious viral and bacterial organisms rapidlymutate and evolve into either more virulent or drug-resistant forms. With increasingly mobile world popula-tions and wide use of anti-viral and anti-bacterial drugsthe stage is set for an accelerated emergence of morevirulent viruses that are resistant to treatment. Examplesinclude SARS, Avian flu, influenza, tuberculosis andmalaria. The possible emergence of Avian flu in Vietnamfurther highlights the urgent need for alternativeapproaches to protection and treatment. For over ahundred years certain physicians have claimed, with noscientific evidence, that low-dose preparations of suchagents can protect and mitigate the effects of infectiousdisease 34. Veterinarians routinely use such agents toinduce herd immunity in agriculture. However, there is apaucity of research and the reports from scientificstudies are mixed. 35, 36 Several double-blind trials on theprevention and treatment of influenza appear to supportsuch claims and call for further investigation. 37 Asmentioned previously, these effects may be due to theimmunostimulatory effects produced by silica and glasscontaminates in the preparations. Other recent researchhas demonstrated that a reduced dose of influenzavaccine can be as effective as higher doses when properlyadministered. 38 More research investigating the possibil-ity of using a RIPT approach to emerging infections isneeded.

Research is required on the low dose induction of thebody’s protective mechanisms to explore the possible useof RIPT on the battlefield, against bioterrorism, and forother purposes. The major challenges in the study ofRIPT include: 1) the need for a sound mechanistic basisfor the study of low-dose stimulation, 2) difficulty inoptimizing low-dose effects, and 3) the failure to exam-ine the entire dose-response range for various toxins. 30

RIPT research could produce an internal “cellular


bioshield” against toxins.

PROPOSED AREAS OF STUDY FOR THECLINICAL UTILITY OF HORMESIS

We propose that a research effort to investigate theclinical utility of hormesis be developed and focus on thefollowing areas.

Toxins. Examination of the protective effects andcellular mechanisms of low-doses in cellularsystems using several types of cell stressors (tox-ins). An initial focus might be toxins with highterrorist potential, including botulinum toxin,cyanide, paraoxon, cadmium, arsenic, con-G andricin.

Viruses. Both tissue culture and animal modelsshould be investigated in an effort to develop lowdose approaches against viral agents includinginfluenza and emerging infectious agents such asAvian flu.

Brain injury. Protection against brain injury due toischemia and neurotransmitter poisoning shouldbe investigated using glutamate and low doses ofsilica. Proteometric and immunostaining methodscould be used to elucidate molecular mecha-nisms.

Cancer. Studies that will screen a variety of chemicalsfor their potential protective effects against cancerin animal and tissue culture models are needed.Effective substances would be further studiedusing genomic and proteometric methods toelucidate mechanisms.

In addition, areas that may involve hormetic dose-responses should be explored.

These include:

Food and Diet. Are the salutogenic effects of low-calorie diets a hormetic response? If so, whatchemicals in foods might account for theseeffects? This area would examine the value oftherapeutic food from the hormetic perspective. 39

Exercise. Are the salutogenic effects of exercise dueto hormesis? If so, we should explore the genomicand proteomic markers that correlate with thiseffect so as to individualize the amount of exerciseoptimal for each individual.

Herbs. Many herbal preparations use low-doses ofmultiple substances to achieve their effects. Arethese effects hormetic in nature?

Homeopathy. Notwithstanding the criticism ofhomeopathy above, many homeopathic sub-stances are not given in ultra-low doses. Thesesubstances may produce hormetic effects at low-doses like herbal preparations.

Stress management. Many stress management

programs involve the application of small inter-mittent “doses” of stressful stimulation as aneffective method for inducing increased toleranceto stress. Calabrese and others have suggested thismay represent a clinical hormetic application. 11

The clinical utility of hormesis is a vast, largely un-charted area, yet the potential clinical implications oflow-dose effects are great. These areas should be investi-gated using rigorous scientific methods in both basicand clinical studies.

REFERENCES1. Southam C, Ehrlich J. Effects of extracts of west-

ern red-cedar heartwood on certain wood-decaying fungi in culture. Phytophathology.1943;33:517-524.

2. Calabrese EJ. Toxicological awakenings: therebirth of hormesis as a central pillar of toxicol-ogy. Toxicol Appl Pharmacol. Apr 1 2005;204(1):1-8.

3. Calabrese E, Baldwin L. Hormesis: a generalizableand unifying hypothesis. Crit Rev Toxicol. July2001;31(4-5):353-424.

4. Jonas W, Kaptchuk T, Linde K. Critical overview ofhomeopathy. Ann Intern Med. 2003;138:393-399.

5. White House Commission of Complementary andAlternative Medicine Policy. Final Report. Wash-ington, DC 2002.

6. Committee on the Use of Complementary andAlternative Medicine by the American Public.Complementary and Alternative Medicine in theUnited States. Washington, DC: Institute ofMedicine; 2005.

7. Rana M, Douglas R. Physics and Chemistry of Glasses.Vol 2; 1961.

8. Stipp D. A little poison can be good for you: thereceived wisdom about toxins and radiation maybe all wet. Fortune. 2003;May 28, 2003(www.fortune.com/fortune/brainstorm/0,15704,454888,00.html)

9. Calabrese E, Baldwin L. Applications of hormesisin toxicology, risk assessment and chemothera-peutics. Trends in Pharmacological Sciences.2002;23:331-337.

10. Calabrese E. Hormetic dose-response relationshipsin immunology: occurrence, quantitativefeatures of the dose-response, mechanisticfoundations, and clinical implications. ClinicalReviews in Toxicology. 2005;35:89-295.

11. Calabrese E, Baldwin L. Hormesis: The dose-response revolution. Annual Rev. Pharmacol.Toxicol. 2003;43:175-197.

12. Calabrese E, Baldwin L. Toxicology rethinks itscentral belief: Hormesis demands a reappraisal

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of the way risks are assessed. Nature. 2003;421:891-892.

13. Calabrese E, Baldwin L. Hormesis as a biologicalhypothesis. Environ Health Perspect. Feb1998;106(Suppl 1):357-362.

14. Damelin L, Alexander J. Metal-induced hormesisrequires cPKC-dependent glucose transport andlowered respiration. Hum Exp Toxicol. July2001;20(7):347-358.

15. Jonas W, Dillner D. Protection of mice fromTularemia infection with ultra-low, serial agitateddilutions prepared from Francisella tularensis-infected tissue. J Sci Explor. 2000;14(1):35-52.

16. Lee Y, Sung F, Lin R, et al. Peripheral blood cellsamong community residents living near nuclearpower plants. Sci Total Environ. Dec 3 2001;280(1-3):165-172.

17. Ives J, Jonas W, Hartings J, et al. Neuroprotectionin experiemntal rat brain injury with ultra highdilution glutamate but not potassium chloride.Soc Neurosci Abst. 2001;27:557.

18. Jonas W, Lin Y, Williams A, Tortella F, Tuma R.Treatment of experimental stroke with low-doseglutamate and homeopathic Arnica montana.Perfusion. 1999;12:452-462.

19. Williams G, Latropulos M. Alteration of liver cellfunction and proliferation: Differentiationbetween adaptation and toxicity. Toxicol Pathol.Jan-Feb 2002;30(1):41-53.

20. Jonas W. The future of hormesis: What is theclinical relevance of hormesis? Crit Rev Toxicol.2001;31:655-658.

21. Rico A. Chemo-defense system. C R Acad Sci III.2001;324(2):97-106.

22. Van Wijk R, Wiegant FA. The similia principle as atherapeutic strategy: a research program onstimulation of self-defense in disordered mam-malian cells. Altern Ther Health Med.1997;3(2):33-38.

23. Jonas W. Directions for research in complementarymedicine and bioterrorism. Altern Ther HealthMed. 2002;8:30-31.

24. Paterson J. Report on Mustard Gas Experiments. JAmer Homoeop Assoc. 1944;37:47-50 and 88-89.

25. Szeto A, Rollwagen F, Jonas W. Rapid induction ofprotective tolerance to potential terrorist agents:a systematic review of low- and ultra-low doseresearch. Homeopathy. 2004;93(4):173-178.

26. Jonas W, Lin Y, Tortella F. Neuroprotection fromglutamate toxicity with ultra-low dose glutamate.NeuroReports. 2001;12:335-339.

27. Marotta D, Marini A, Banaudha K, et al. Non-lineareffects of cycloheximide in glutamate treatedcultured rat cerebellar neurons. Neurotoxicology.2002;23:307-312.

28. Ives J, Moffett J, Peethambaran A, et al. Potential

involvement of glass-derived silicates in "homeo-pathic neuroprotection". Proc Natl Acad Sci.Currently Under Review.

29. Linde K, Jonas WB, Melchart D, Worku F, WagnerH, Eitel F. Critical review and meta-analysis ofserial agitated dilutions in experimental toxicol-ogy. Hum Exp Toxicol. 1994;13(7):481-492.

30. Calabrese E, Baldwin L. Tales of two similarhypotheses: The rise and fall of chemical andradiation hormesis. Hum Exp Toxicol. Jan2000;19(1):85-97.

31. Mallick P, Mallick J, Guha B, Khuda-Bukhsh A.Ameliorating effect of microdoses of apotentized homeopathic drug, ArsenicumAlbum, on arsenic-induced toxicity in mice. BMCComplement Altern Med. Oct 22 2003;3(1):7.

32. Gaddipati J, Rajeshkumar N, Grove J, et al. Low-dose cadmium exposure reduces human pros-tate cell transformation in culture and up-regulates metallothionein and MT-1G mRNA.Nonlinearity Biol Toxicol Med. 2003;1(2):199-212.

33. Delbancut A, Barouillet M, Cambar J. Evidenceand mechanistic approach of the protectiveeffects of heavy metal high dilutions in rodentsand renal cell cultures. Signals and Images.Dordrecht/London: Kluwer Academic Publish-ers; 1997:71-82.

34. Frye J. Public comment from the National Center forHomeopathy. Rockville, MD: White House Com-mission on Complementary and AlternativeMedicine Policy; Feb. 22, 2002 2002.

35. Taylor S, Mallon T, Green W. Efficacy of a homeo-pathic prophylaxis against experimental infec-tion of calves by the bovine lungworm Dictyocau-lus viviparus. Veterinary Record. 1989;124:15-17.

36. Day C. Clinical trial in bovine mastitis. Br Homeop J.1986;75:11-14.

37. Vickers A, Smith C. Homeopathic Oscillococcinumfor preventing and treating influenza andinfluenza-like syndromes. Cochrane Database SystRev. 2004.

38. Belshe R, Newman F, Cannon J, et al. Serumantibody responses after intradermal vaccinationagainst influenza. N Engl J Med.2004;351(22):2286-2294.

39. Brandt K, Christensen L, Hansen-Moller J, et al.Health promoting compounds in vegetables andfruits: a systematic approach for identifying plantcompounds with impact on human health.Trends in Food Science and Technology. 2004;15:384-393.


THE MANAGEDIMMUNE SYSTEM:PROTECTING THEWOMB TO DELAYTHE TOMB

Rodney R. Dietert and Michael S. Piepenbrink

Department of Microbiology and Immunology

College of Veterinary Medicine

Cornell University

Ithaca, NY 14853

Phone: 607 253-4015

Fax: 607 253-3384


Key Words: Immune balance, Skewed responses,

Perinatal development, Environmental management

ABSTRACT

It is likely to come as a surprise to most lay audiencesthat much of our safety net for ensuring protectionagainst the adverse effects of chemicals in the environ-ment or drugs is based on evaluations performed largelyusing adult animals. Of course in the case of drugs,clinical trials are also required, but again this usuallymeans testing for unanticipated and undesired sideeffects on adult human populations. Certainly, multi-generation studies are performed to test chemical safetylevels, but these usually require chemicals or drugs toinduce profound teratogenic (disruption of earlydevelopment) or reproductive alterations to trigger aconcern. The potential jeopardy lies with more subtleeffects of chemicals on non-adults that could neverthe-less adversely impact health. So in extrapolating safetydata derived largely from adults, we have presumed tounderstand and fully accept the spectrum of risks tonon-adults.

Yet, where the human experiment has been carried outon the largest scale, as in the case of fetal exposure toalcohol or environmental tobacco smoke, we know thatthe fetus is particularly sensitive (1-3). The effects ofthese agents on the fetus are not fully predicted basedon similar adult exposures, nor do they have identicalconsequences in terms of dose response and persistenceof effects. The bottom line is that the amount of alcohol

required for adverse effects on a young adult woman ismuch different than the amount that can impact thefetus if exposure occurs during specific vulnerableperiods of gestation. So does this mean we have takenthis lesson to heart?

As a society, we expend countless dollars, Euros, etc. onvarious micronutrients, dietary supplements and nutri-tive compounds that we hope will encourage our im-mune systems to perform better against the latest viral orbacterial infection crossing our path at that time. Butthis “pop a pill-immediately lose the ill” approach tobetter health masks a broader issue concerning optimumimmune health over the course of a lifetime, a coursemost of us hope is long indeed. The question must beraised whether the science is now in place or at leastaccumulating to encourage a more comprehensive andlasting approach to managing the immune system withthe individual and our most sensitive sub-populations inmind.

The recent comprehensive review of hormesis and theimmune system (4) suggests that it is not simply life-stagebased immunotoxicity where there is a need to improveour understanding and predictability of outcome.Indeed, understanding immunomodulatory exposuresand the range of doses that impact the immune systemby age and gender is the broader goal. This article willdiscuss the possible benefits arising from early attentionto immune health and the application of fundamentaldevelopmental immunology to the neonatal, adolescent,adult and geriatric stages of life.

Immune Balance

Over a lifetime of health challenges, we face the need tomount robust immune responses against a variety ofinfectious agents and potential cancer cells. We arepresented with wildly different classes of viruses, bacte-ria, parasites and tumor cells each with their own hostdefense challenges. Additionally, it is important torespond to vaccinations with effective, immediateresponses and robust immunological memory to ensureadequate long-term protection. To meet such challenges,it is helpful to have effective immune balance among thedistinct segments of the immune system that affordprotection against different categories of disease-induc-ing agents. Today’s infection is not necessarilytomorrow’s health challenge

While immune balance is a potential paradigm topromote life-long health, historically, it has not been onthe radar screen. This is particularly evident when onerealizes that drugs and herbs promising immune en-hancement may not be the universal panacea desiredeither in the individual or across a population. Certainlya better understanding of dose-response toxicity,hormetic responses and genetic variation must be pairedwith intended immunomodulation. Immune enhanced

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relief from viral infections in one person might meanincreased risk of autoimmune reactions in another. So“enhancement” is not necessarily effective balance andenhancement for one person may mean disease foranother.

The Paradigm Shift: Immune Skewing vs. Immunosup-pression

Historically, immunotoxicologists focused on profoundgeneralized immunosuppression as the primary concern.It was relatively easy to identify toxicants that at relevantdoses caused atrophy of the thymus or significantpopulation loss within the spleen. With the increasedincidence of atopy, asthma, and certain autoimmunediseases in recent years, researchers are now focused ontargeted immunosuppression and targeted immune-mediated damage resulting from autoimmune or inflam-matory responses. Changes associated with thesedifferent health risks can be subtle involving little to noloss of immune cells or histopathological alterations toimmune tissues. The goal then is to avoid imbalance andeither the lack of certain required responses or thepromotion of responses that are undesirable and mightcompromise host tissue integrity. But emphasizingimmune balance as the ultimate goal requires a differentmind set and potentially new approaches for immuneassessment. It also requires the recognition that across aheterogeneous human population, individuals start atdifferent places on an immune balance curve based ongenetic heterogeneity. Therefore, the one-recipe-for-allapproach has obvious pitfalls and should immediately beviewed with considerable skepticism.

The importance of immune balance and the potentialmechanistic bases for skewing were discussed in severalrecent reviews (5,6). The precise mechanisms andpathways for controlling T helper 1 (Th1) vs. T helper 2(Th2) responses among dendritic cells and T lympho-cytes are still the subjects of active ongoing research.One thing is very clear from these reviews. While it ischallenging to dissect all of the components influencingimmune balance in a fully matured adult, it is a com-pletely different set of issues to consider immune bal-ance within a moving target, as is the case with thedeveloping immune system. The following sectionillustrates this point as well as the important windows ofopportunity or, in some cases, vulnerability that appearto exist.

The Immune Maturation Dilemma

The developing fetus presents a remarkable paradox interms of immune cell differentiation, functional develop-ment, and tissue integrity (i.e. homeostatic) consider-ations. For example, the T cell system needs to matureefficiently to meet the challenges presented by theneonatal environment at birth; but during the course ofmaturation, the potential for maternal-fetal allogeneic

reactions must be minimized. One hallmark of preg-nancy is a strong skewing toward Th2 cytokines, whichenables the survival of the fetus in the genetically-dissimilar (i.e. allogeneic) maternal environment (7).Placental trophoblasts seem to help determine the Th2nature of the maternal-fetal interface. When these cellsexpress Th1 cytokines and receptors such as interferon-gamma and its receptor, there is an increased risk ofpreeclampsia, a pregnancy complication also calledtoxemia and characterized by high blood pressure,swelling, and fever (8). Using a rodent model, Sefrioui etal. (9) showed that the Th1 vs. Th2 cytokine pattern inutero was critical in determining whether tolerance orimmunity developed. Leibnitz (10) recently discussedthe fact that, at birth, humans have a T cell system that isreduced in Th1 functional capacity. This is the capacitythat is necessary for effective anti-viral defense but alsopromotes tissue/organ rejection. Aggressive Th1 func-tion, in utero, would jeopardize maternal-fetal compatibil-ity and increase the risk of pregnancy complications.Evidence for the reduced Th1 capacity of newborns isreflected in the fact that the neonate is severely reducedin the production of interferon-gamma, the hallmarkTh1 cytokine (11). For the neonate to thrive, develop-ment of Th1 must proceed at a rapid pace shortly afterbirth. Transplantation studies suggest that the newbornis far more accepting of transplants than a child sixmonths or more of age because it is essentially Th1deficient compared to an adult (12).

All of this leads to the conclusion that the late fetus andearly newborn have a highly skewed immune capacitycompared with the final balance that will be obtained inmost juveniles and adults. In effect, desired immunebalance will only exist in the newborn if and when theTh1 capacity matures effectively. Clearly, our attentionneeds to be directed at those environmental factors (e.g.diet, toxicants, infections, and vaccinations) and therelevant doses that allow the fetus to thrive but alsopromote much needed Th1 maturation immediatelyfollowing birth. Conversely, we need to identify factorsand dose ranges that have the capacity to delay and/orimpair neonatal Th1 function.

Windows of Opportunity/Vulnerability

Given that the late fetus and early newborn lacks thedesired immune balance necessary to meet later lifedisease challenges, this represents a window in whichattention to environment and immune maturation islikely to reap life long rewards. Delayed or impairedTh1 maturation would be expected to leave the indi-vidual with a potentially life-long immune imbalance. Incontrast, promotion of optimum Th1 capacity immedi-ately after birth would increase the likelihood of effectiveviral vaccine responses, reduce the risk of childhoodasthma and atopy and potentially influence the risk oflater life cancer. Considering the major differencesbetween the adult and fetal immune systems and the


different strategies needed to achieve immune balancein each, it is hardly surprising that adult safety data haveonly minimal application to the fetal/early neonatal lifestage. The inherently skewed immune system of thefetus and newborn provides an ideal opportunity fordirect evaluation of environmental conditions thatpromote effective juvenile immune balance vs. those thatinterfere with this outcome. However, to accomplish thiswe will need directly applicable dose response dataincluding knowledge of potential hormetic responsesrather than information extrapolated from the adultimmune system.

Furthermore, the opportunity to truly manage theimmune system may best be directed toward the early(non-adult) stages of development. Developmentalimmunotoxicity results, to date, suggest that in utero andearly neonatal effects tend to be more persistent thanthose induced in adults (13). So the idea that oneshould attempt late-life corrections to an unbalancedimmune system (skewed at birth) when adult healthproblems arise may be too little too late. The early-managed immune system is simply the most efficient andcost effective approach in terms of a reducing a lifetimeof health care costs.

Early Environment-Later Life Disease?

One question surrounding the concept of increasedsensitivity of the developing immune system to environ-mental modulation is the question of evidence for actualimpact on human health. Because non-teratogenicimmune changes would result in a greater risk of disease,the challenge is to look at epidemiological patterns inhuman disease in concert with mechanistic animalstudies. Altered patterns of disease associated withimmunomodulation is a matter of discerning populationshifts imposed on an already existing incidence ofdisease. Perhaps the most readily accessible examplesconcern the increased incidence in asthma, atopy andcertain autoimmune diseases. For this mini-review,disease consideration will be restricted to childhoodasthma.

According to a recent Pew Foundation report (14), theincident of asthma in industrialized counties has in-creased dramatically in recent decades. This has resultedin a significant public health cost. In 2002, there wereapproximately 16 million adolescents with asthma (15).The impact of the increase has been dramatic. Forexample, there were 14 million missed school days in1996 up from 6.6 million in 1980 (16). The environmen-tal causes for this increase are certainly multi-factorialand involving various toxins, infectious agents, and air-born pollutants, the latter that can exacerbate symptoms.However, there is one common theme in terms of thetype of immunomodulatory change contributing toincreased incidence. That change is a shift in immunebalance toward a Th2 bias (17). In fact, Anderson (17)

argues that it is the retention of the fetal immuneimbalance that is the hallmark of early asthma. Thetenet has also been advanced by Bousquest et al. (18)who discuss the likelihood that fetal-expressed genespromoting Th2 may continue to be inappropriatelyexpressed in some neonates increasing the risk ofasthma. Additionally, maternal immune responsesduring pregnancy appear to have the potential to helpshape the subsequent Th profile of the neonate (19).One perinatal indicator of subsequent risk of asthmaappears to be the serum immunoglobulin E levels ofcord blood at birth (20).

In the newborn, those environmental factors thatpromote strong early life Th1 responses tend to beprotective against childhood asthma while those thatimpair effective Th1 maturation seem to contribute toelevated incidence of the disease. Several studies fromEurope have indicated that exposure of the newborn tobarnyard environments (including endotoxin-ladenbacteria) was highly protective against childhood asthma(21,22). These results are compatible with the conceptthat stimulating Th1 cytokines and responses at keyperiods of early development can help effective immunebalance to be achieved. On the reverse side of thisequation, exposure to environmental contaminants thatimpair Th1 functional development could leave adoles-cents with an increased risk of asthma. Based on theirimmunotoxic effect, certain heavy metals such as leadwould be candidates to promote the risk of asthma viathe persistent Th2 bias they can create (23-26).

Dietary intake, both maternally and in the neonate, isimportant in immune balance. Early malnourishmentseems to allow Th2 polarization to be maintained (27).Vitamin A deficiency can also contribute to Th2 skewing(28). In contrast, consumption of dietary nucleic acidsappears to skew the profile toward Th1 (29). Glu-tathione levels in antigen presenting cells seem to helpsteer responses toward Th1 or Th2. Depletion ofglutathione depresses Th1 responses skewing the re-sponse in favor of Th2 (30).

Genetics can also play a role overlaying the environmen-tal exposure issue. Among farming-family children, thegenotype of an important cell surface receptor forimmune signaling, Toll-like receptor-2 (TLR-2), influ-enced the risk of asthma (31). Another factor influenc-ing Th1/Th2 balance as well as risk of asthma appears tobe the cytotoxic T-lymphocyte antigen 4 (CTLA-4) geneand its polymorphisms in the human population (32).This suggests that segments of the human population arelikely to respond differently when exposed to specificenvironmental risk factors based on the genetics of theimmune cell signaling as well as their inherited immunebalance capacities. Such genotype-by-environmentinteractions indicate the challenges in conductingappropriate analyses that have the statistical power toassess changes within sub-populations. Futhermore, it

10 BELLE Newsletter

suggests the importance of employing animal modelsthat can mimic potentially vulnerable human sub-populations. Given this genetic heterogeneity, we shouldnot expect positive or adverse risk factors to shift morethan a segment of the human population across animmune balance boundary associated with clinicalsymptomology.

In summary, early onset asthma has increased in inci-dence with environmental factors playing a major role inthis increase. Current evidence involving infectiousagents, environmental toxins, dietary factors and geneticpolymorphism can be placed within a unified immuno-logical concept. It suggests that managing the fetal andneonatal immune system to reduce persistence of thefetal (Th2-skewed) immune phenotype and to promoterapid and effective Th1 maturation has the potential tosignificantly reduce the risk of asthma across the popula-tion.

Hormesis and the Managed Immune System

The recent review of hormesis and the immune systemby Calabrese (4) has important implications for themanaged immune system. This review summarizedseveral key points. First, the numerous examples ofhormesis pertaining to immune responses indicates theimportance of identifying beneficial and adverse effectsassociated with different portions of dose-responsecurves. Rather than blanket labeling environmentalfactors as beneficial or adverse in terms of immunemanagement, we need to tailor effective and beneficialdoses to each individual (based on genotype). Thesecond message from this review is that virtually all of thehormesis examples described in the literature arerestricted to the adult immune system or adult-derivedcells examined in vitro. This does not mean thathormesis is not key to the developing immune system.Rather, it reflects the extreme dearth of comprehensivedose response comparisons that exist for differentwindows of immune development. If we err by univer-sally applying adult safety results to the fetus, then wealso must be cautious about applying hormetic doserange across age groups. Obviously, direct age-baseddata are needed to optimally manage the early immunesystem.

CONCLUSIONS

Attention to the early immune system and those environ-mental factors and dose ranges that promote effectiveimmune balance is a cost-effective approach to reducinglater life disease risk and health care costs. Applicationof adult safety data is severely limited for this purpose.Instead, the use of direct exposure information leadingto timely and effective neonatal T helper 1 maturationappears key to long-term immune balance. While focuson the early immune system has not been a traditionalsafety approach, it seems clear that this is when an

immune imprint becomes established; one that impactsgreatly on postnatal health and well being.

REFERENCES1. Barber, K., Mussin, E., and Taylor, D.K. Fetal

exposure to involuntary maternal smoking andchildhood respiratory disease. Annals of AllergyAsthma and Immunology 76: 427-430, 1996.

2. Windham, G.C., Bottomley, C., Birner, C., andFenster, L. Age at menarche in relation tomaternal use of tobacco, alcohol coffee and teaduring pregnancy. American Journal of Epide-miology 159: 862-871, 2004.

3. Gauthier, T.W., Ping, X.D., Harris, F.L., Wong, M.,Elbahesh, H., and Brown, L.A. Fetal alcoholexposure impairs alveolar macrophage functionvia decreased glutathione availability. PediatricResearch 57: 76-81, 2005.

4. Calabrese, E.J. Hormetic does-response relation-ships in immunology: occurrence, quatitationfeatures of the dose response, mechanisticfoundations, and clinical importance. CriticalReviews in Toxicology 35(2/3): 89-295, 2005.

5. Romagnani, S. Immunologic influences on allergyand the Th1/Th2 balance. Journal of Allergyand Clinical Immunology 113: 395-400, 2004.

6. Pulendran, B. Variegation of the immune re-sponse with dendritic cells and pathogen recog-nition receptors. Journal of Immunoogy 174:22457-2465, 2005.

7. Bjorksten, B. The intrauterine and postnatalenvironments. Journal of Allergy and ClinicalImmunology 104: 1119-1127, 1999.

8. Bannerjee, S., Smallwood, A., Moorhead, J.,Chambers, A.E., Papageorghiou, A., Campbell,S., and Nicholaides, K. Placental expression ofinterferon-gamma (IFN-gamma) and its receptorIFN-gamma R2 fail to switch from early hypoxicto late normotensive development in preeclamp-sia. Journal of Clinical Endocrinology andMetabolism 90: 944-952, 2005.

9. Sefrioui, H., Donahue, J., Gilpin, E.A., Srivastava,A.S., and Carrier, E. Tolerance and immunityfollowing in utero transplantation of allogeneicfetal liver cell: the cytokine shift. Cell Transplant12: 75-82, 2003.

10. Leibnitz, R. Development of the human immunesystem. In: Holladay, S.D. (Ed.) DevelopmentalImmunotoxicology. CRC Press, Inc. Boca Raton,FL. 2005. pp 21-42.

11. Holt, P.G., Clough, J.B., Holt, B.J., Baron-Hay, M.J.,Rose, A.H., Robinson, B.W., and Thomas, w.R.Genetic “risk” of atopy is associated with delayedpostnatal maturation or T cell competence.Clinical and Experimental Allergy 22: 1093-1099,


1992.

12. Morrow, W.R. and Chinnock, R.E. Survival afterheart transplantation. In Tejani, A.H., Harmon,W.E. and Fine R.N. (Eds.) Pediatric Solid OrganTransplantation. Munksgaard, Copenhagen,Denmark, 2000, pp 417-426.

13. Luebke R., Chen, D., Dietert, R., King, M., Yang,Y., and Luster, M . Increased sensitivity of thedeveloping immune system to xenobiotics:experimental evidence supporting the conceptof developmental immunotoxicity testingguidelines. Journal of Toxicology and Environ-mental Health, in press. 2005.

14. Pew Foundation, Attack Asthma: Why Americaneeds a public health defense system to battleenvironmental threats. 2000.

15. United States Centers for Disease Control andPrevention. Morbidity mortality weekly report 53(#7) February 27, 2004.

16. United States Centers for Disease Control andPrevention. Surveillance for Asthma: UnitedStates, 1980-1999. Morbidity and mortalityweekly report 251 (SS01): 1-13. 2002

17. Anderson, G.P. The immunobiology of earlyasthma. Medical Journal of Australia 177 (SupplS): 47-49, 2002.

18. Bousquest, J., Jascot, w., Yssel, H., vignola, A.M.,and Humbert, M. Epigenetic inheritance offetal genes in allergic asthma. Allergy 59: 138-147, 2004.

19. Herz, U., Ahrens, B., Scheffold, A., Joachim, R.,Radbruch, A., and Renz, H. Impact of in uteroTh2 immunity on T cell deviation and subse-quent immediate-type hypersensitivity in theneonate. European Journal of Immunology 30:714-718, 2000.

20. Sadeghnejad, A., Karmaus, W., David, s.,Kurukulaaratchy, J., Matthew, S and HasanArshad, S. Raised cord serum immunoglobulinE increases the risk of allergic sensitization atage 4 and 10 and asthma at age 10. Thorax 59:936-942, 2004.

21. Von Ehrenstein, O.S., Von Mutius, E., Illi, S.,Baumann, l., Bohm, O., and Von Kreis, R.Reduced risk of hay fever and asthma amongchildren of farmers. Clinical and ExperimentalAllergy 30: 187-193, 2000.

22. Riedler, J., Braun-Fahrlander, C.H., Eder, W.,Schreuer, M., waser, M., Maisch, Exposure tofarming in early life and development of asthmaand allergy: a cross-sectional survey. Lancet 358:1129-1133, 2001.

23. McCabe, M. J. Jr, and Lawrence D.A. Lead, amajor environmental pollutant, isimmunomodulatory by its differential effects onCD4+ T cell subsets. Toxicology and Applied

Pharmacology 111: 13-23, 1991.

24. Miller, T.E., Golemboski, K.A., Ha, R.S., Bunn,T.L., Sander, F.S. and Dietert, R.R. Developmen-tal exposure to lead causes persistentimmunotoxicity in Fisher 344 rats. ToxicologicalSciences 42: 129-135, 1998.

25. Synder, J.E, Filipov, N. M., Parsons, P.J., andLawrence, D.A. The efficiency of maternaltransfer of lead and its influence on plasma IgEand splenic cellularity of mice. ToxicologicalSciences 57: 87-94, 2000.

26. Dietert, R. R., Lee, J-E., Hussain, I., andPiepenbrink, M. Developmentalimmunotoxicology of lead. Toxicology andApplied Pharmacology 198: 86-94, 2004.

27. Neyestani, T.R., and Woodward, B. Bllod concen-tration of Th2 immunoglobulins are selectivelyincreased in weanling mice subjected to acutemalnutrition. Experimental Biology and Medi-cine 230: 128-134, 2005.

28. Stephensen, C.B., jiang, X., and Freytag, T. Vita-min A deficiency increases the in vivo develop-ment of IL-10-positive Th2 cells and decreasesdevelopment of Th1 cells in mice. J Nutr 134:2660-2666, 2004.

29. Sudo, N., Aiba, Y., Oyama, N., Yu, XN, Matsunga,M., Koga, Y and Kubo, C. Dietary Nucleic acidand intestinal microbiota synergistically promotea shift in the Th1/Th2 balance toward Th-1skewed immunity. Int Arch Allergy Immunol135: 132-135, 2004.

30. Peterson, J.D., Herzenberg, L.A., and Vasquez, K.Gluathione levels in antigen-presenting cellsmodulate Th1 versus Th2 response patterns.Proceeding of the National Academy of SciencesUSA 95: 3071-3076, 1998.

31. Eder, W., Klimecki, W., Yu, L., von Mutius, E.,Riedler, J., Braun-Fahtlander, C., Nowak, d.,Martinex, F.D., and the ALEX Study Team. Toll-like receptor 2 as a major gene for asthma inchildren of European farmers. Journal ofAllergy and Clinical Immunology, 113: 482-488,2004.

32. Munthe-Kass,, M.C., carlson, K.H., Helms, P.J.,Gerritsen, J., Whyte, M., Feijen, M.,Skinningsrud, B., Main, M., Kwong, G.N., Lie,B.A., Lodrup, Carlson, K.C., and Undlien, D.E.CTLA-4 polymorphisms in allergy and asthmaand the TH1/Th2 paradigm. Journal of Allergyand Clinical Immunology 114: 280-287, 2004.

12 BELLE Newsletter

LOW-DOSERADIATION AND ITSCLINICALIMPLICATIONS:DIABETES

Guan-Jun Wang 1, Xiao-Kun Li 2, Kazuo Sakai 3 and Lu

Cai 1,2,4,*

1 Department of Hematology and Oncology

The First University Hospital

Jilin University Medical College

Changchun 130021

P.R. China

2 Department of Biopharmacy

Collage of Pharmacy

and Biopharmaceutical Research & Development Center

Jinan University

Guangzhou 510080

P.R. China

3 Low Dose Radiation Research Center

Central Research Institute of Electric Power Industry

2-11-1 Iwado-Kita, Kome

Tokyo, 201-8511

4 Departments of Medicine, Pharmacology and

Toxicology,

and Radiation Oncology

the University of Louisville School of Medicine

Louisville 40202

USA

Corresponding author at Department of Medicine

University of Louisville

511 South Floyd Street

MDR 533, Louisville

KY 40202

Phone: 502-852-5215

Fax: 502-852-6904


ABSTRACT

Induction of hormesis and adaptive response by low-doseradiation (LDR) has been extensively indicated. Adaptiveresponse induced by LDR was not only resistant todamage caused by subsequently high-dose radiation, butalso cross resistant to other non-radiation challengessuch as chemicals. Mechanisms by which LDR inducesthe preventive effect on radiation- or chemical-inducedtissue damage include induced or up-regulated expres-sion of protective proteins such as heat shock proteinsand antioxidants. Since oxidative damage to tissues is amajor pathogenesis of many human diseases includingdiabetes, this review will summarize available data withan emphasis on the preventive effect of LDR on thedevelopment of diabetes and the therapeutic effect ofLDR on diabetic cardiovascular complications. Theavailable data indicated that pre-exposure of mice toLDR reduced the incidence of alloxan-induced diabetes,and also delayed the onset of hyperglycemia in diabetes-prone non-obese diabetic mice. Experiments withanimals indicated the therapeutic effect of low-intensityor power laser (LIL or LPL) radiation on skin woundhealing, which has stimulated clinical use of LIL to cureskin ulcer in diabetic patients. Mechanisms by whichLDR prevents diabetes, though they are unclear now,may include the induction of pancreatic antioxidants toprevent β cells from oxidative damage and immuno-modulation to preserve pancreatic function. For LILtherapeutic effects on diabetic wound healing, mecha-nisms may include its antioxidant action, immuno-modulation, cell-proliferation stimulation as well asimprovement of systemic and wound-regional microcir-culation. Therefore, although there are only a fewstudies indicating LDR prevention of the development ofdiabetes, many studies have demonstrated LDR, specifi-cally LIL, therapeutic effectiveness of diabetic woundhealing. These preliminary results encourage furtherassessment of the clinical implications of LDR to diabe-tes-related areas.

Key words: Low-dose radiation, hormesis, adaptiveresponse, diabetes, diabetic complications

INTRODUCTION

Low-dose radiation (LDR)-induced hormesis has beenextensively studied for the last two decades (Luckey1982). It includes stimulation of DNA, RNA, and proteinsynthesis as well as DNA repair activity, increase incellular antioxidant capacity, prolongation of life spanand activation of immune functions (Cai 1999; Calabrese2002; Calabrese and Baldwin 2003). These cellularhormetic effects contribute to a protection of cells invitro or in vivo against gene mutation, DNA damage, andchromosome aberrations caused by subsequent radiationor toxic chemicals, which was called an adaptive re-


sponse (Olivieri et al., 1984; Cai and Liu 1990; Cai et al.,1994; Cai and Jiang 1995; Cai and Wang 1995; Cai andCherian 1996).

Since extensive scientific evidence shows the stimulationof immunological function, antioxidant activity and DNArepair ability, an important issue is whether LDR-inducedhormesis or adaptive response can be manipulated formedical and other benefits, as discussed six years ago in aspecial issue of Belle 1999 (Cai et al., 1999) and ad-dressed recently by Dr. Calabrese and his associates(Calabrese and Baldwin 2002; Calabrese 2004). Inclinical implication, LDR has been introduced as aneffective therapy of non-Hodgkin's lymphoma (Richaudet al., 1998; Kennerdell et al., 1999; Girinsky et al., 2001).For instance, in the study by Richaud et al., (1998), mostpatients treated by LDR showed efficient response, inparticular for follicular lymphoma. They lived withoutany acute nonlymphoblastic leukemia or myelodysplasicsyndrome with a median follow-up of 88 months. Al-though they did not discuss any issues related to LDR-induced adaptive response or hormesis for the mecha-nism of this beneficial effect, this could not exclude therole of LDR hormesis in immunity and adaptive responsein the hematopoietic tolerance in this efficient treat-ment. In addition, since Alzheimer's disease (AD) isrelated to oxidative damage to neurons leading to cellloss and LDR enhances brain antioxidant activity(Kojima et al., 1999; Yamaoka et al., 2002), whether LDR-enhanced activity of antioxidants in brain could protectbrain cells from oxidative damage to prevent AD hasbeen discussed. Human epidemiological study showed alow incidence (4.39%, 25/570) of AD in the populationof high natural radiation background area as comparedto that (4.95%, 25/505) of control population (Wei et al.,1996). Therefore, the similar mechanisms of LDR-induced hormesis and adaptive response may be alsoclinically implicated for prevention or therapy of otherdisorders.

Diabetes mellitus has dramatically increased globally, andaffects 18 millions of Americans (Bardsley and Want2004). Type 1 diabetes is a lack of insulin production andType 2 diabetes is predominantly due to resistance to theeffects of insulin. Both Type 1 and Type 2 have the samelong-term complications including skin ulcer (Bardsleyand Want 2004). Type 1 diabetes occurs because theinsulin-producing cells of the pancreas (called β cells)are destroyed by the body's own immune system forunknown reasons. There are other people who developa condition similar to type 1 diabetes - characterized bydestruction of the β cells but without autoimmunereaction, as the streptozotocin (STZ)- or alloxan (ALX)-induced diabetes in animal models (Shafrir 1990). Type2 diabetes is the most common form of diabetes, and itscause is more complex. In type 2 diabetes, high bloodglucose arises despite an initial abundance of the hor-mone insulin. With progression of the disease they candevelop a deficiency of insulin similar to people with

type 1 diabetes.

Oxidative stress is now known to be involved in almost ofall pathological states of pancreatic β-cells either in Type1 or Type 2 diabetes (Shafrir 1990; Haskins et al., 2003).Oxidative stress is characterized by increased productionof reactive oxygen or nitrogen species (ROS or RNS)such as superoxide, hydrogen peroxide, nitric oxide andperoxynitrite and/or decreased concentrations ofantioxidants and antioxidant enzymes including glu-tathione (GSH), vitamin E, ascorbate, glutathioneperoxidase (Gpx), superoxide dismutases (SOD), andcatalase. The β cell destruction by ROS and/or RNS,whether induced by STZ or ALX given exogenously orelicited by cytokines, is a process that occurs throughboth apoptotic and necrotic mechanisms. After diabetesonset, the secondary oxidative stress caused by diabetichyperglycemia, hyperlipidemia and even inflammationalso play a critical role for most diabetic complications(Rosen et al., 2000; Cai and Kang 2001).

Therefore, this review assessed the available data withemphasis on: (1) Effects of LDR on the development ofdiabetes; (2) Effects of LDR on diabetic complications;and (3) Possible mechanisms by which LDR prevents thedevelopment of diabetes and diabetic complications.Finally, the clinical diabetes-related implications of LDRare discussed.

EFFECTS OF LDR ON THEDEVELOPMENT OF DIABETES

Takehara et al. (1995) performed the first investigationof the effects of LDR on ALX-induced diabetes in the ratmodel. ALX caused a significant degranulation of β cellsin the pancreas along with an increase in fasting bloodglucose. These changes were significantly prevented byLDR. The preventive effect of LDR on the developmentof diabetes was further supported by subsequent experi-ments (Yamaoka et al., 1996; Takahashi et al., 2000; Sakaiet al., 2004). These studies indicate the following fea-tures:

LDR prevented ALX-induced diabetes in a rat model andalso in a spontaneously developed non-obese diabetic(NOD) mouse model (Takehara et al., 1995; Takahashi etal., 2000). LDR which induced the protection against thedevelopment of diabetes could be acute exposure(Takehara et al., 1995; Takahashi et al., 2000) and also bechronic exposure (Sakai et al., 2004). For acute exposurewith gamma rays, among doses of 0.25, 0.5, 1.0 and 2.0Gy used, only exposure to 0.5 Gy provided the significantprotection against ALX-induced diabetes (Takehara etal., 1995). There was no significant difference for use ofone or two doses of 0.5 Gy gamma-rays in the protectiveeffect against the development of diabetes (Takahashi etal., 2000). However, the time interval between LDR anddiabetes onset seems an important factor to determine

14 BELLE Newsletter

the protective effect against the development of diabetes.As shown in Figure 1, non-LDR treated NOD micedeveloped diabetes starting at 15 wks of age (panel A)and the incidence of diabetes was 60% (panel B), whileLDR treated NOD mice at 12, 13 or 14 wks of agedeveloped diabetes starting at 3 to 7 wks later and alsoshowed low incidence of diabetes. The most effectiveprotection was noted in the group of mice irradiated byLDR at 13 wk of age.

The decreased blood glucose and increased plasmainsulin suggest a protection of LDR against β-cell dam-age in NOD diabetic mice (Takahashi et al., 2000).Examination of β cells performed for the untreatedNOD mice and LDR-treated mice (irradiated at 13 weeksof age) showed a significant increase in apoptotic β celldeath in the pancreases of untreated NOD mice wasevident, but not in the LDR-treated NOD mice. Thisobservation is keeping with other studies indicating thatapoptosis of pancreatic β cells are responsible for thedevelopment of diabetes in NOD mice (Kurrer et al.,1997; Nakayama et al., 2002).

In addition to the protective effect of LDR against thedevelopment of diabetes, LDR was found to provide atherapeutic effect on diabetic hyperglycemia. RecentlySakai et al. (2004) investigated the effects of chronic LDRon C57BL/KsJ-db/db mice with Type 2 diabetes. Thesemice develop Type 2 diabetes by 10 weeks of age, due toobesity, and are characterized by hyperinsulinemia. Agroup of 10-week old female mice was irradiated for lifeat dose rate of 0.7 mGy/hr. The urine glucose levels ofall of the mice were strongly positive at the beginning ofthe irradiation. In the LDR-treated diabetic mice,however, a decrease in the urine glucose level wasobserved in three mice, one in the 35th week, one in the52nd week and one in the 80th week. No recovery fromthe diabetes was observed in the 12 mice of non-LDR-irradiated diabetic group. These preliminary resultssuggest that LDR provides a therapeutic effect ondiabetes.

EFFECTS OF LDR ON DIABETICCOMPLICATIONS

The major pathogenic cause for various diabetic compli-cations is attributed to diabetes-overproduced ROS/RNSand diabetes-impaired antioxidants in tissues (Rosen etal., 2000; Cai and Kang 2001). Whether LDR is able toprevent or cure various complications of diabetesmellitus is a very interesting issue. Although there was nostudy directly using low-dose ionizing radiation, therewere several studies using low levels of non-ionizingirradiation such as laser radiation and magnetic field.

Ionizing radiation

In an early study by Yamaoka and Komoto (1996),indications for treatment at the Misasa Hot Spring, a

radon producing radioactive spring, diabetes patientsalong with other patients with pain and hypertensionwere noted. The hot-spring treatment with radon wasfound to significantly improve the vasodilation andalleviated diabetic symptoms. In the animal studies byTakahashi et al. (2000) and Sakai et al. (2004), LDR notonly decreased the incidence and percentage of develop-ing diabetic mice, but also enhanced the surviving rateof these type 1 and type 2 diabetic animals. For instance,at the age of 90 weeks animal survival was 75 % in theLDR-treated NOD mice and only 40 % in the non-LDR-treated NOD mice. Mortality was delayed and thehealthy appearance was prolonged in the irradiated miceby about 20-30 weeks compared with non-LDR-treatedNOD mice. For the study using chronic LDR, survivalrates of both LDR-treated and non-LDR-treated diabeticmice were 100% at 30 weeks of age (these mice devel-oped diabetes at 10 weeks of age), but 33% in LDR-treated diabetic mice and 0% in non-LDR-treateddiabetic mice at 120 weeks of age (Sakai et al., 2004).Although these indications are not direct parameters ofdiabetic complications, these indications may result fromthe protective effects of LDR against diabetes-initiatedvarious complications.

Non-ionizing radiation

Low-intensity or –power laser (LIL or LPL) radiation hasbeen extensively used for therapy of diabetic patients. Inrecent years, LIL has gained considerable recognitionand importance among treatment modalities for variousmedical problems including wound repair processes,musculoskeletal complications and pain control (Reddy2003). In diabetic conditions, LIL successfully enhancedwound healing, tensile strength and systemic or localmicrocirculation in the diabetic animal models (Stadleret al., 2001; Reddy 2003; Byrnes et al., 2004; Kawalec etal., 2004) and diabetic patients (Schindl et al.,1997,1998,1999a,b,2002; Zinman et al., 2004).

Although different kinds of lasers have been used for thistherapy, the efficient therapy depends on the wavelengthof the electromagnetic radiation (Stadler et al., 2001;Reddy 2003; Kawadec et al., 2004). For instance, theability of photostimulation to promote healing ofimpaired wounds using a Ga-As laser in rats with STZ-induced diabetes was compared with the effects of a He-Ne laser. Reddy (2003) found that the He-Ne laserappears to be superior to the Ga-As laser in promotingwound healing in diabetic animal models.

Low-power electromagnetic field was also used for atherapeutic purpose. Chebotar'ova and Chebotar'ov HIe(2003) investigate the effectiveness of combinations oflow-power electromagnetic therapy and low-powervariable magnetic field on the clinical-electroneuromyography in 12 patients with diabeticpolyneuropathies. Exposure to both provided significantdecreases in neurological deficit and required insulin


daily dose and significant increases in nerve conductionvelocity, the muscle compound action potentials (musclepower) and peripheral outflow in some patients.

POSSIBLE MECHANISMS FOR LDRPREVENTIVE AND THERAPEUTICEFFECTSLDR-enhanced endogenous antioxidant activity

Oxidative stress is the critical factor responsible fordiabetic onset and complications, while antioxidants canprevent both diabetic onset and complications (Cai andKang 2001; Rosen et al., 2001). Development of diabetesinduced either by T cell-mediated inflammatory autoim-mune reaction or chemicals such as STZ and ALX islargely attributed to ROS and RNS formation leading toβ-cell destruction (Fig. 2). In addition, low concentra-tions of antioxidants exists in animal pancreatic isletsalso make it vulnerable to oxidative damage (Lenzen etal., 1996; Tiedge et al., 1997). There is evidence indicat-ing that up-regulation of SOD made mice resistant todiabetes development (Kubisch et al., 1997).Thioredoxin (Trx), a redox (reduction/oxidation)-active protein, protects cells from oxidative stress.Transgenic mice with specific expression of Trx inpancreatic islets showed a significantly lower incidence ofspontaneously developed and STZ-induced diabetes ascompared to their wild-type counterparts (Hotta et al.,1998).

LDR significantly increases endogenous antioxidants indifferent tissues including liver, spleen, brain and testes(Yamaoko et al., 1991,1998,1999,2002,2004a; Kojima etal., 1998,1999; Zhang et al., 1998). The activation and/orinduction of antioxidants include SOD, GSH, Gpx,glutathione reductase (GR), catalase and Trx. In thesetissues, the increased antioxidants by LDR significantlyprevents tissue damage from various oxidative stresses,for instance, cardon tetrachloride-induced liver damage(Kojima et al., 1998; Yamaoka et al., 2004a), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine- or Fe-NTA-inducedbrain damage (Kojima et al., 1999; Yamaoka et al., 2002),and radiation-induced testis damage (Zhang et al.,1998). In studies by Takehara et al. (1995) and Takhashiet al. (2000), LDR (0.5 Gy) also significantly increasesthe SOD activity in the pancreas of non-diabetic mice.The activity of pancreatic SOD in ALX-induced or NODdiabetic mice was significantly decreased, but thisdecrease could be prevented by LDR. In addition, inALX-induced diabetic mice, plasma and pancreatic lipidperoxide levels were also significantly increased, but notin LDR-irradiated diabetic mice (Takehara et al. 1995).These results suggest that the increased antioxidantcapacity of pancreases by LDR is one of the majormechanisms to prevent ALX-induced or spontaneouslydeveloped diabetes (Fig. 2).

For the mechanisms of the attenuation of diabetic

hyperglycemia and complications in diabetic patientswho received radon hot spring, Yamaoka et al (2004b)found that the radon therapy enhanced the antioxidantfunctions, such as the activities of SOD and catalase,along with inhibition of lipid peroxidation and totalcholesterol produced in the body, although the therapyalso increased the levels of plasma insulin and glucose-6-phosphate dehydrogenase. This study further supportsthe concept that LDR protects against diabetes and itscomplications probably through induction of antioxi-dants.

LDR-modulated immune function

Type 1 diabetes loses the ability to produce insulin dueto the destruction of the insulin-producing β cells in thepancreas. The β cells are targets of an autoimmuneattack during which the diabetic's own immune cells,especially the T cells, recognize unique proteins in the βcells as foreign and go about ridding the β cells from thepancreas. Like other autoimmune diseases, type 1diabetes can be caused by defects in the primitive cellsthat are the precursor cells of the blood and immunesystem called hematopoietic stem cells (HSC). HSCs arecapable of producing the entire set of cells that com-prises the immune system, as well as red blood cells andplatelets. To support of the importance of normalhematopoietic system for maintaining a normal immunefunction to avoid the development of type 1 diabetes,bone marrow transplantation was shown to halt autoim-mune processes and reverse the damage caused byautoimmune cells in NOD mice (Li et al., 1996; Chiltonet al., 2004; Nikolic et al., 2004). We have demonstratedthat LDR can stimulate HSC proliferation and mobilizesHSCs into peripheral circulation (Li et al., 2004). ThisHSC stimulating and mobilizing effect of LDR may beone of the mechanisms to maintain a normal immunefunction to avoid the development of Type 1 diabetessuch as in the case of NOD mice (Takahashi et al., 2000).

Ina and Sakai (2004) have shown the possibility for LDRto ameliorate severe autoimmune diseases. In theirexperiment, chronic low-dose-rate γ irradiation at 0.35 or1.2 mGy/h was found to significantly prolong the lifespan of MRL-lpr/lpr mice carrying a deletion in theapoptosis-regulating Fas gene that markedly shortens lifedue to severe autoimmune disease. Immunologicalmodifications were indicated by a significant increase ofCD8+ T cells and a significant decrease of CD3+ CD45R/B220+ as well as CD45R/B220+ CD40+ cells, along withamelioration of total-body lymphadenopathy, splenom-egaly, proteinuria, and kidney and brain syndromes. Inanother clinical study in which an attenuation of diabetichyperglycemia and complications were observed in thediabetic patients received radon hot spring (Yamaoka etal., 2004b), radon-got spring significantly increasedpatient’s immune response such as enhanced concanava-lin A-induced mitogen response. Therefore, LDR-modulated immune function may be one of the mecha-

16 BELLE Newsletter

nisms underlying LDR prevention of type 1 diabetes (Fig.2).

Multiple mechanisms involved in LIL improvement ofdiabetic wound healing

The mechanisms discussed above are predominantly forthe preventive or therapeutic action of low-dose ionizingradiation. However, we do not exclude their roles in thetherapeutic effect of LIL on diabetic wound healingsince LIL acts as similar function in certain aspects(Wilden and Karthein 1998). Small amount of ROS/RNS generation is one of the factors responsible forLDR- induced hormesis and adaptive response (Cai1999; Cai et al., 1999). Exposure of cells to LIL alsogenerates certain amounts of ROS/RNS (Callaghan etal., 1996; Vladimirov et al. 2004), which may stimulateadaptive mechanisms, shown by increased antioxidantactivities (Karageuzyan et al., 1998; Fujimaki et al., 2003;Kao and Sheen 2003).

Besides antioxidant action of LIL, the directly stimulat-ing cell proliferation and procollagen synthesis throughactivation of signaling pathways are also importantmechanisms for the diabetic wound healing (Duan et al.,2001; Pereira et al., 2002; Schindl et al., 2003; Vinck et al.,2003). Whether stimulating HSCs by LIL is also involvedin the enhancing effect on diabetic wound healingremains unclear; however, direct exposure of woundtissues to growth factors promoted wound healingprocesses of diabetic animals (Galeano et al., 2004a,b),and basic fibroblast growth factor was found to beincreased in the wound tissue of diabetic rats in responseto LIL (Byrnes et al., 2004).

A specific mechanism for LIL to enhance diabetic woundhealing may be the improvement of systemic and woundregional microcirculation (Schindl et al., 1998,2002). Forinstance, in the patients with diabetic microangiopathyreceiving a single LIL irradiation, skin blood circulation,by means of temperature recordings detected by infraredthermography, significantly increased as compared tonon-LIL treated diabetic patients.

PROSPEROUS REMAKERS

Radiation is known to have significant effects on livingorganisms dependent on the dose received. At highdoses, radiation destroys cells in tissue. At low doses, onthe other hand, radiation is no longer considered to beas harmful as once thought. Hormesis and adaptiveresponse of cells or tissues in response to LDR wereextensively documented (Luckey 1982; Cai 1999; Cai etal., 1999; Calabrese 2002; Calabrese and Baldwin 2002).However, debates for the induction and importance ofhormetic effects and adaptive responses still exist, inparticular for the risk of LDR in genetic instability andcarcinogenesis (Johansson 2003; Poumadere 2003;

Calabrese 2004). Among the several issues raised in thesedebates, one is how the adaptive response or hormesisinteracts with the bystander effect in determiningbiological responses at low doses of radiation and theshape of the dose-response relationship (Zhou et al.,2003). Although the epidemiological and experimentalstudies remain not strong enough to change the currentpolicy of radiation safety, the phenomenon of LDR-induced hormesis and adaptive response could not benegative. Definitively large epidemiological studies andmore detail experimental studies with new and accuratetechniques remain required before determining whetherthe current linear non-threshold model needs to bechanged.

However, clinical application of LDR is a differentsituation. The targets of clinical application with LDRwould be mainly patients with various disorders. It isclear that none of the medications used in clinics isabsolutely non-toxic. Therefore, evaluation of LDR for itsclinical application should be also realistic and parallelas evaluation of other new medications. If LDR can playa critical approach in prevention or therapy of certaindisorders, such as diabetes, we should not ignore it.

This short review collected the available data demonstrat-ing possible implications for the prevention of diabetesdevelopment and therapy of diabetic complications.These preliminary results showed that pre-exposure ofanimals to LDR significantly prevented ALX-induced orspontaneously developed diabetes. Although there wasno experiments of using low-dose ionizing radiation tocure any diabetic complications, clinical observationimplied a therapeutic effect of low-dose radon ondiabetic complications in the patients. More importantly,effective therapeutic effect of LIL on skin wound healingof diabetic subjects has attracted the attention of investi-gators and clinicians. Although exact mechanisms bywhich LDR prevents the development of diabetes andprovides the therapeutic effect on diabetic complicationremain largely unknown, several possibilities may beincluded, such as the increase in antioxidants, immuno-modulation, HSC stimulation and peripheral mobiliza-tion, stimulation of target cells and improvement ofsystemic and wound-regional microcirculation as out-lined in Figure 2. Therefore, further experiments on thistopic remain required, and we believe that LDR implica-tions in diabetes-related areas is an important area ofresearch.

ACKNOWLEDGEMENTS

The work cited in this review is supported in part byresearch grants from Philip Morris USA, Inc. andAmerican Diabetes Association to LC.

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20 BELLE Newsletter

Figure 1. LDR prevention of the development of diabetes in NOD mice. Pane A represents the time point at whichthe first mouse from the groups treated with LDR (0.5 Gy) at 12, 13 or 14 weeks (w) of age and without LDR (controlgroup) spontaneously developed diabetes (hyperglycemia). Results indicate that the first mouse developed diabetes isat 22 weeks of age in the group of mice with LDR at 13 weeks of age (13 w), which is 7 weeks later than that(15 weeksof age) in control group. Panel B represents the incidence of diabetic mice in different groups. Results indicate that10 % of mice with LDR at 13 weeks of age (13 w) developed diabetes at 24 weeks of age, which is much lower than

those in the control and other LDR-treated groups. Figures were made by the authors, based on the results from thestudy by Takahashi et al. (2000).


Figure 2. Outlines of the possible mechanisms underlying LDR’s protective effect against the development of diabetesand therapeutic effect on diabetic complications. IR: ionizing radiation; NIR: non-ionizing radiation. Solid line and

dash line indicate the increasing and decreasing effect, respectively.

22 BELLE Newsletter

BREAST CANCER,CHEMOTHERAPYAND HORMESIS

Lorne J. Brandes, MD

Departments of Medicine

and Pharmacology/Therapeutics

University of Manitoba

and Section of Hematology/Oncology

CancerCare Manitoba

675 McDermot Avenue

Winnipeg MB R3E0V9

Canada

Phone: 204-787-2139

Fax: 204-786-0196


ABSTRACT

N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine(DPPE; tesmilifene) is a novel antihistaminic andchemopotentiating agent that has a hormetic effect onDNA synthesis in MCF-7 human breast cancer cells invitro and stimulates the growth of experimental tumorsin rodents. In a prospectively randomized phase threetrial (NCIC MA.19), 152 patients who were co-adminis-tered DPPE and doxorubicin survived 50% longer(P<0.03) than 153 patients who were administered thesame dose and schedule of doxorubicin alone. Atclinically relevant in vitro concentrations that do notinhibit the P-glycoprotein (P-gp) pump, DPPE selectivelysensitizes cancer cells that express the multiple drugresistance (MDR+) phenotype, making them moresusceptible to the cytotoxic effects of chemotherapeuticagents, including anthracyclines and taxanes. Based onits previously demonstrated interaction with histamine atCYP3A4, a P450 that metabolizes arachidonic acid, andits induction of high levels of PGI

2 in the gut of rodents,

modulation by DPPE of the intracellular concentrationof arachidonate products, such ashydroxyeicosatetraeinoic acids (HETEs), implicated inincreased cancer cell proliferation and metastasis, ispostulated.

I. Treatment of metastatic breast cancer (MBC)

Historically, the systemic treatment of metastatic breastcancer (MBC) has been predicated on achieving acytotoxic effect to shrink tumors [1]. Anti-hormonaltherapies, such as tamoxifen and aromatase inhibitors,

are generally preferred in the treatment of estrogen-receptor (ER)-positive breast cancer in the metastaticsetting, especially in older women [2]. The use ofchemotherapy is reserved for the approximately fiftypercent of pre- or post-menopausal women who havemore aggressive tumors that are generally not hormon-ally-responsive, or in cases where anti-hormonal agentsfail to control the disease.

II. Chemotherapy and survival in MBC

Despite the availability of highly potent cytotoxic drugs,including anthracycline derivatives (doxorubicin andepirubicin) [3] and taxanes (paclitaxel and docetaxel)[4], significant improvement in survival is rarely ob-served in metastatic breast cancer, even though majortumor responses (shrinkage of 50% or more in size) andan increase in the time to progression (TTP; the periodfrom starting on treatment until disease progression) areoften observed. A minority of patients with aggressivebreast cancer whose tumors overexpress type 2 humanepidermal growth factor (Her-2) [5], benefit from theaddition of the specific Her-2 antibody, trastuzumab(Herceptin), to taxane therapy, resulting in increased (≤25%) survival [6].

III. Increased survival in MBC when DPPE is co-adminis-tered with doxorubicin

In a prospective randomized international phase threestudy (MA.19) conducted by the National CancerInstitute of Canada Clinical Trials Group, 152 womenwith metastatic breast cancer who received the combina-tion of doxorubicin and the experimentalchemopotentiating drug, N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine (DPPE;tesmilifene), survived 50% longer than 153 women whoreceived the same dose and schedule of doxorubicinalone (23.6 months vs. 15.6 months, respectively; p<0.03)[7]. Contrary to earlier phase two studies [8,9], theoverall survival benefit in DPPE-treated patients resulteddespite the absence of an improvement in tumor re-sponse rates or TTP.

An independently conducted sub-group analysis (unpub-lished) revealed that a significant survival benefit of theDPPE/doxorubicin combination occurred mainly inpatients with a short (≤ 36 months) disease-free interval(DFI; the time from diagnosis to disease recurrence) andthose with ER-negative tumors, both surrogates foraggressive disease [10]. Recent in vitro studies (unpub-lished) suggest a selective chemopotentiating effect ofclinically relevant concentrations of DPPE on cancercells expressing the multiple drug resistant (MDR+)phenotype, common in aggressive cancer [11].

Based on the results of MA.19, a confirmatory phasethree international study (YMB 1002-02) in 700 womenwith metastatic breast cancer is currently comparing the


combination of DPPE, epirubicin and cyclophosphamideto the same dose and schedule of epirubicin and cyclo-phosphamide alone in patients with DFI ≤ 36 months,stratified for ER status (positive or negative). Theprimary endpoint of this new pivotal study is survival;secondary endpoints are TTP, response rates and qualityof life. Accrual is expected to be completed by late 2005,with survival outcome known as early as late 2006.

IV. Pharmacology of DPPE

A. DPPE binding to “antiestrogen binding sites” on P450enzymes

Although its mechanism(s) of action remain to be fullyelucidated, the pharmacological profile of DPPE mayprovide important clues to its chemopotentiating action.A diphenylmethane derivative that resembles tamoxifen,DPPE lacks the stilbene bridge and third phenyl ringnecessary to bind the ER [12]. The drug was synthesizedin an effort to determine a biological role forantiestrogen binding sites (AEBS) that also bindtamoxifen [13]. As opposed to the ER, which is locatedprimarily in the nucleus in breast and uterine tissue, theAEBS is located in the microsomal fraction, derived fromendoplasmic reticulum, and is found in most tissues,although most abundantly in liver [14]. Becausetamoxifen binds to both sites, an assessment of a putativerole for AEBS in its antiestrogenic/antitumor actioncould only be discerned with a selective agent.

Radioligand binding studies revealed DPPE does notbind to the ER in rat uterine cytosol, but is equipotent totamoxifen to bind the AEBS in rat liver microsomes [12].DPPE also antagonizes the uterotropic effects of exog-enous estrogen in vivo, suggesting a role for AEBS in theantiestrogenic action of tamoxifen [15]. Spectral analysisassessing DPPE binding to purified human isozymesultimately led to the determination that the AEBSrepresents the “substrate” site on certain microsomalcytochromes P450, including CYP3A4, CYP2D6 andCYP1A1 [16].

The 3A4 isozyme is important in the metabolism ofarachidonic acid, lipids, hormones and many drugs [17],including antineoplastic agents such as taxanes, vincaalkyloids, etoposide and cyclophosphamide/ifosphamide. Substrates for CYP3A4 are usually alsosubstrates for the co-regulated P-glycoprotein (P-gp)membrane pump [18]. When overexpressed in cancercells, P-gp contributes to multiple drug resistance [19].High (50µM) concentrations of DPPE have been shownto inhibit P-gp in vitro, overcoming drug resistance topaclitaxel [20].

B. Histamine and proliferation: Interactive binding ofDPPE and histamine at P450

The structure of DPPE is also similar to H1-antihista-

mines such as diphenhydramine and hydroxyzine, but ismuch weaker than these agents in classical assays of H

1-

activity that measure antagonism of histamine-inducedmuscle contraction [21]. In contrast, DPPE competesmore strongly than conventional H

1 and H

2 antagonists

for the binding of 3H-histamine to microsomal AEBS/P450, an interaction that correlates with cytotoxicity tobreast cancer cells in vitro [22]. Histamine binding toAEBS/P450 results from its affinity for the heme ironmoiety of the enzymes [16]. Through binding to theP450 substrate site, “AEBS” ligands such as DPPE,tamoxifen and many other arylalkylamines [23] stericallyhinder histamine binding to the heme iron.

A role for intracellular histamine in promoting normaland malignant growth was originally proposed byKahlson and Rosengren [24]. P450 enzymes are alsoimportant regulators of cell function, including prolif-eration [25], and may be overexpressed in malignantcells of breast origin [26]. We have recently identified anin situ histamine/P450 complex in rat liver microsomes[27], and amplified upon Kahlson’s original hypothesiswith the suggestion that histamine may modulate growthby regulating the catalytic activity of various P450s[16,27].

At non-cytotoxic concentrations that correlate with itsinhibition of 3H-histamine binding to P450, DPPEinhibits DNA synthesis in mitogen stimulated normalspleen cells in vitro [28]. In contrast, DPPE exerts ahormetic (biphasic) effect on DNA synthesis in trans-formed and malignant cells, killing them at high concen-trations in vitro [28]. DPPE stimulates tumor growth inmice (L5178Y lymphoma) and rats (DMBA-inducedmammary carcinoma) [29]. Correlating with this find-ing, systemic administration of DPPE markedly increasesinflammation and mitosis in rat skin painted with thetumor promoting phorbol ester, PMA [29].

C. L-histidinol and DPPE: Dissimilar agents with similareffects on histamine binding, DNA synthesis,cytoprotection and chemopotentiation

Warrington [30] showed that L-histidinol, the alcoholprecursor to histidine (that, in turn, may be converted tohistamine by histidine decarboxylase) [31], potentiateschemotherapy to effect cures in experimental murinetumors, while protecting the bone marrow. In collabora-tive studies with Warrington [28], L-histidinol wasdemonstrated to compete for 3H- histamine binding inmicrosomes, to inhibit DNA synthesis in normal mitogenstimulated spleen cells and to have a biphasic effect onDNA synthesis in transformed and malignant cells. LikeL-histidinol, DPPE was observed to protect the bonemarrow of mice from high doses of daunorubicin and 5-fluorouracil, while increasing tumor-free survival in miceadministered doxorubicin [28]. In addition to itsprotection of the bone marrow, a cytoprotective effect ofDPPE in the gut of rats exposed to cold-restraint stress

24 BELLE Newsletter

has been linked to the induction of a 10-fold increase inlevels of prostacyclin (PGI

2) [32].

V. DPPE sensitization of MDR+ cells appears to beindependent of P-gp inhibition

At clinically relevant concentrations (3-5 µM), signifi-cantly lower than those required to inhibit the P-gp [33],DPPE appears to selectively potentiate chemotherapydrugs in MDR+ cells in vitro. This finding suggests somealternative, possibly novel, mechanism by which DPPEovercomes drug resistance. Warrington previouslyreported a similar profile for L-histidinol in overcomingMDR [34].

VI. Hormetic effect on DNA synthesis andchemopotentiation by DPPE may be linked to themodulation of arachidonate metabolism

The observation that DPPE causes (i) low-concentrationstimulation and high-concentration inhibition of DNAsynthesis in MCF-7 breast cancer cells in vitro; (ii)stimulation of inflammation and mitotic activity inphorbol-painted skin [29];(iii) growth-stimulation of rodent tumors; and (iv)induction of high levels of PGI

2 in the gut [32], raises the

possibility that, like its cytoprotective effects,chemopotentiation by DPPE may be linked to its interac-tion with P450s and/or lipoxygenases involved inarachidonate metabolism. An interaction with theseenzymes might result in an increase or decrease incellular levels of various lipids and eicosanoids that,depending on their concentration, stimulate or inhibitDNA synthesis.

That only cells expressing the MDR+ phenotype appearto be sensitized to chemotherapy by DPPE could belinked to their increased turnover of arachidonate. Forexample, aggressive cancers with increased metastaticpotential appear to have significantly higher levels ofprostaglandins [35]. Thus, co-administration of DPPEmay “set up” high grade MDR+ cells for killing, not byinhibiting the P-gp, but by causing further significantperturbations (up or down) in certain eicosanoids.Candidates might include hydroxyeicosatetraenoic acids(HETEs). High levels of 5- and 12-(S)-HETE are impli-cated in increased mitosis and tumor aggressivity [36],while inhibition of their synthesis leads to apoptosis ofbreast cancer cells [37]. Thus, the hormetic effect ofDPPE on DNA synthesis in transformed and malignantcells, possibly important to its chemopotentiation, maybe mediated indirectly by the modulation of HETEs orother lipid/eicosanoid products that stimulate/inhibitcell division and affect cell survival.

CONCLUSION

Validation of a survival benefit with the addition of DPPEto epirubicin and cyclophosphamide will have important

implications for the treatment of humans with aggressiveforms of breast cancer. Chemopotentiation of otherantineoplastic drugs by DPPE in patients with variousrefractory cancers has been reported [33], includingcyclophosphamide in patients with androgen-indepen-dent prostate cancer [38]. These findings raise the hopethat DPPE may have the potential to increase survival inmany types of chemotherapy-responsive cancer.

Statement of potential conflict of interest:

Dr. Brandes is the inventor of DPPE (tesmilifene) andcould benefit financially from its successful commercial-ization. DPPE is licensed by the University of Manitobato YM Biosciences, Mississauga, ON.

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9. Khoo K, Brandes LJ, Reyno L et al. Phase II trial ofN,N-diethyl-2-[4-(phenylmethyl)


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10. Chevallier B, Mosseri V, Dauce JP, Bastit P, Julien JP,Asselain B. A prognostic score in histologicalnode negative breast cancer. Br J Cancer 1990;61: 436-440.

11. Thomas H, Coley HM. Overcoming multidrugresistance in cancer: an update on the clinicalstrategy of inhibiting p-glycoprotein. CancerControl 2003; 10: 159-165.

12. Brandes LJ, Hermonat MW. A diphenylmethanederivative specific for the antiestrogen bindingsite found in rat liver microsomes. BiochemBiophys Res Commun 1984; 123: 724-728.

13. Sutherland RL, Murphy LC, Foo MS, Green MD,Whybourne AM, Krozowski ZS. High affinityantiestrogen binding site distinct from theestrogen receptor. Nature 1980: 288: 273-275.

14. Watts CK, Sutherland RL. High affinity specificantiestrogen binding sites are concentrated inrough microsomal membranes of rat liver.Biochem Biophys Res Commun 1984; 120: 109-115.

15. Brandes LJ, Hogg GR. Study of the in vivoantiestrogenic action of N,N-diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine. HCl(DPPE), a novel intracellular histamine antago-nist and selective antiestrogen binding siteligand. J Reprod Fert 1990; 89: 59-67.

16. Brandes LJ, Queen GM, LaBella FS. N,N-diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine (DPPE), achemopotentiating and cytoprotective agent inclinical trials: Interaction with histamine atcytochrome P450 3A4 and other isozymes thatmetabolize antineoplastic drugs. CancerChemother Pharmacol 2000; 45; 298-304.

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19. Ling V. Multidrug resistance: molecular mechanismsand clinical relevance. Cancer ChemotherPharmacol 1997; 40 (Suppl): S3-8.

20. Menendez AT, Raventos-Suarez C, Fairchild C et al.Mechanism of action of DPPE, achemosensitizing agent. Proc Amer AssocCancer Res 1998; 39: 3462 (abstract).

21. Kroeger EA, Brandes LJ. Evidence that tamoxifen is ahistamine antagonist. Biochem Biophys Res

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22. Brandes LJ, Bogdanovic RP, Cawker MD, LaBella FS.Histamine and growth: interaction ofantiestrogen binding site ligands with a novelhistamine site that may be associated withcalcium channels. Cancer Res 1987; 47: 4025-4031.

23. LaBella FS, Brandes LJ. Enhancement of tumorgrowth by drugs with some common molecularactions. Mol Carcinogen 1996; 16: 68-76.

24. Kahlson G, Rosengren E. Histamine formation asrelated to growth and protein synthesis. In:Biogenic Amines as Physiological Regulators(Blum JJ, Ed.) Englewood-Cliffs NJ, Prentice-Hall 1970; pp 223-238.

25. Nebert DW. Proposed role of drug-metabolizingenzymes: Regulation of steady state levels of theligands that affect growth, homeostasis, differen-tiation and neuroendocrine functions. MolEndocrinol 1991; 5: 1204-1214.

26. Reiger MA, Ebner R, Bell DR, Kiessling A et al.Identification of a novel mammary-restrictedcytochrome P450, CYP4Z1, with overexpressionin breast carcinoma. Cancer Res 2004; 64: 2357-64.

27. Brandes LJ, Queem GM, LaBella FS. Displacement ofhistamine from liver cells and cell componentsby ligands for cytochromes P450. J Cell Biochem2002; 85: 820-824.

28. Brandes LJ, LaBella FS, Warrington RC. Increasedtherapeutic index of antineoplastic drugs incombination with intracellular histamine antago-nists. J Natl Cancer Inst 1991; 18: 1329-1336.

29. Brandes LJ, Beecroft WA, Hogg GR. Stimulation of invivo tumor growth and phorbol ester-inducedinflammation by N,N-diethyl-2-[4-(phenylmethyl) phenoxy] ethanamine. HCl, apotent ligand for intracellular histamine recep-tors. Biochem Biophys Res Commun 1991; 179:1297-1304.

30. Warrington RC. A novel approach for improvong theefficacy of experimental cancer chemotherapyusing combinations of anticancer drugs and L-histidinol. Anticancer Res 1986; 6: 451-464.

31. Moya-Garcia AA, Medina MA, Sanchez-Jimenez F.Mammalian histidine decarboxylase: fromstructure to function. Bioessays 2005; 27: 57-63.

32. Glavin GB, Gerrard JM. Characterization of thegastroprotective effects of N,N-diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine. HCl, anon-H1, non-H2 histamine antagonist. Digestion1990; 47: 143-148.

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26 BELLE Newsletter

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34. Warrington RC, Fang WD. Reversal of the multidrug-resistant phenotype of Chinese hamster ovarycells by L-histidinol. J Natl Cancer Inst 1989; 81:798-803.

35. Rolland PH, Martin PM, Jacquemier J, Rolland AM,Toga M. Prostaglandin in human breast cancer:Evidence suggesting that an elevated prostaglan-din production is a marker of high metastaticpotential for neoplastic cells. J Natl Cancer Inst1980; 64: 1061-70.

36. Honn KV, Tang DG, Gao IA et al. 12-Lipoxygenasesand 12(S)-HETE: role in cancer metastasis.Cancer and Metastasis Rev 1994; 13: 365-396.

37. Tong W-G, Ding, X-Z, Adrian TE. The mechanisms oflioxygenase inhibitor-induced apoptosis inhuman breast cancer cells. Biochem Biophys ResCommun 2002; 296: 942-948.

38. Brandes LJ, Bracken SP, Ramsey EW. N,N-diethyl-2-[4-(phenylmethyl) phenoxy]ethanamine. HCl incombination with cyclophosphamide: an activelow toxicity for metastatic hormonally unrespon-sive prostate cancer. J Clin Oncol 1995; 13: 1398-1403.

28 BELLE Newsletter

Starting January 2005 the journal NON-LINEAR-ITY: Biology, Toxicology and Medicine(www.nonlinearity.net), now entering its third year,will be editorially directed by BELLE, published andowned by the University of Massachusetts/Amherst.The consolidation of all journal activities under theauspices of BELLE is designed to enhance both thevisibility and leadership role of BELLE in the areaof low dose biological effects as well as to facilitatean improved promotion of the journal and a moredirect interaction amongst contributing authors,BELLE and the scientific community.NONLINEARITY in Biology, Toxicology and

GOALA growing number of scientists, including

toxicologists, pharmacologists, biostatisticians,epidemiologists, occupational and environmentalmedical researchers and others have begun todisplay considerable interest in the topic ofhormesis, a dose response phenomenon character-ized by a low dose stimulation and a high doseinhibition. While there are many professionalsocieties that have a general interest in dose re-sponse relationships, none explicitly is devoted tothe topic of understanding the nature of the doseresponse in general and hormesis in particular. Thediversity of professional societies that may considerdose response issues, including hormesis, is nonethe-less quite broad ranging from the agricultural to thebiomedical and clinical sciences. However, nearlywithout exception, these societies tend to be stronglyorganized around professional advancement and notfocused on specific scientific concepts. This makesthe issue of hormesis one of diffuse interest across abroad range of professions. The present situationrepresents a major obstacle for the integratedassessment of the dose response in general andhormesis in particular. In order to provide intellec-tual and research leadership on the topic ofhormesis, a new professional association has beencreated called the International Hormesis Society(IHS).

The Society will be dedicated to the enhance-ment, exchange and dissemination of ongoingglobal research efforts in the field of hormesis. Inaddition, the Society will also strongly encourage theassessment of the implications of hormesis for suchdiverse fields as toxicology, risk assessment, risk

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