biological eflects of inhaled minerals gnoncn d. gurnnrn, jn. · 2007. 8. 30. · iarity with...

American Mineralogist, Volume 77, pages 225-243, 1992

Biological eflects of inhaled minerals

Gnoncn D. Gurnnrn, Jn.Geology/Geochemistry, MS D469, Los Alamos National laboratory, Los Alamos, New Mexico 87545, U.S.A.

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Numerous studies present data on the biological effects of inhaled minerals, but thesedata are often disseminated within reports that primarily address the asbestos minerals.Furthermore, these reports are normally published in journals that are unfamiliar to mostminerals scientists. This review compiles these data in order to facilitate an understandingof the known biological effects of minerals. An introduction to the types of studies fromwhich the data were drawn is given so that those unfamiliar with such studies can assessthe data critically.

In general, minerals exhibit a range ofbiological activities from apparently inactive orslightly active, such as hematite, to highly fibrogenic and carcinogenic, such as fibrousbrucite ("nemalite"). The zeolites also exhibit such a range, with some mordenite beingslightly active and erionite being highly active; however, erionite is the only zeolite thathas been studied extensively.

Although several mechanisms have been proposed to explain how minerals induce dis-ease, it is still unclear why minerals exhibit a range in biological activity. The diversity ofmineral species holds great potential for probing these mechanisms, especially when min-eralogical data are integrated with biological data. Unfortunately, many of the studiesreporting data on the biological effects of inhaled minerals fail to report detailed miner-alogical information; hence, it is difficult at present to interpret the biological activities ofminerals in terms of their physical and chemical properties. More collaboration betweenminerals scientists and health scientists would benefit this area ofresearch by enabling anintegration of mineralogical and biological data. Important mineralogical data that areonly rarely considered in biological research include exact mineral content of the specimen(i.e., identification and abundance of contaminants), physical and chemical properties ofminerals, and surface properties of minerals.

IutnonucrroN asbestos minerals. Nevertheless, a wide range of fibrousand nonfibrous minerals has been studied to some extent

Because of their potential to induce a number of lung for potential health hazards, although such data are fre-diseases (e.g., fibrosis, lung cancer, and mesothelioma), quently hidden within reports that focus on asbestos.the asbestos minerals, fibrous serpentine (chrysotile) and These data are important, however, for several reasons.fibrous amphiboles, have been the focus of numerous ex- First, all humans are exposed to mineral dusts from bothperimental studies, governmental regulations, and exten- anthropogenic and natural sources. For example, in a 45-sive public concern. Early studies ofriebeckite-asbestos yr study on the mineral contents oflungs from residentsminers (Wagner et al., 1960) revealed an association be- of Tokyo, Shishido et al. (1989) found that by the earlytween exposure to riebeckite asbestos and mesothelioma, 1980s over 800/o of Tokyo's residents had been exposeda rare cancer with an extremely high morbidity rate. Sub- to mineral dusts. Similar observations have been madesequently, a higher than expected incidence of mesothe- in a variety ofurban environments, and nonoccupationallioma was found in American asbestos workers who were exposure to mineral dusts can also result from living inexposed primarily to chrysotile (Selikoffet al., 1964, 1965) a rural environment (e.g., S6bastien et al., 198 l, I 984).butalsotoamphiboles(Ross, I984),whichoccurredmore Second, various minerals are used both as replace-frequently in the lungs of these workers (Langer and No- ments for asbestos and in numerous other commerciallan, 1989). The widespread use of the asbestos minerals products, and the health risks associated with these min-meant that a large population was exposed and poten- erals may differ from those associated with serpentinetially at risk, and so began the proliferation of research asbestos or with amphibole asbestos. If minerals are toon the biological effects ofasbestos. be used and regulated properly, it is important to assess

Fear of asbestos exposure led to a replacement of the accurately the risk from exposure to each mineral andasbestos minerals with substitutes and a skewing of the not simply assume that all fibrous minerals are equallyresearch on the biological effects of minerals toward the hazardous.0003404x/92/0304422s$02.00 22s

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Third, the mechanisms by which minerals in general(including asbestos) are toxic and carcinogenic can be morereadily elucidated if differences in the toxicity and car-cinogenicity of various minerals can be related to fun-damental differences in crystal structure and crystalchemistry. Collaborative efforts between minerals scien-tists and life scientists would be extremely effective atachieving this. Unfortunately, very few minerals scien-tists are involved directly in such research, primarily be-cause of scientific language barriers and a lack of famil-iarity with current issues in health-related mineral-dustresearch.

In order to address the latter problem, this paper willreview the health risks associated with a variety of min-erals. Ross ( 198 l, 1984) reviewed the human health haz-ards associated with the asbestos minerals in a form ac-cessible to geoscientists, and Mossman et al. (1990)presented a review ofthe current issues in this researchfield. These papers are useful starting points for anyoneinterested in the extensive literature available on asbes-tos. Likewise, the research on the the biological effects ofsilica is extensive, and Heppleston (1984) provides anintroduction to this literature. However, no such com-pilation exists for the literature on numerous other fine-grained minerals. The intentions of this paper are (l) tocompile the available data on the biological effects of clays(exclusive of chrysotile), zeolites, and several other fine-grained minerals; (2) to facilitate a better understandingof the data by introducing the research techniques usedin the studies from which the data were drawn; and (3)

to provide a foundation that can be used by mineralsscientists interested in pursuing this area of research.

The intentions ofthis paper are to avoid any discussionof mineral regulation or the regulatory implications of thematerial reviewed. Though many of the minerals coveredin this review are toxic, carcinogenic, or both in sometests, the risk to humans exposed under normal condi-tions may be minimal. As will be noted, many of theexperiments used to assess the pathogenicity of a mineralinvestigate the mechanisms of pathogenicity or evaluatethe pathogenicity of a specific mineral relative to otherminerals. These experiments do not necessarily emulatetypical conditions of human exposure; hence the resultsmay not reflect the exact response that would be expectedin humans. The assessment of risk from exposure to aspecific mineral is an extremely involved task. The inter-ested reader is directed to the article by Mossman et al.(1990) for an introduction to the problem and a list ofpertinent references. Current federal regulations, includ-ing those for minerals, can be found in Codes of FederalRegulations (CFR 29, part 1910.1000). CfR 29 is revisedannually on July I and is found in most libraries.

INrnolucrroN To BIoLocrcAL REPToRTS

Determination of a substance's biological activity

Exposure to mineral dusts has been linked with a va-riety of lung diseases. Exposure to a fibrogenic mineral

GUTHRIE: EFFECTS OF INHALED MINERALS

can result in fibrosis (production of scar tissue) in the

lung, which can impair the function of the lung. Exposure

to a tumorigenic (or carcinogenic) mineral can result in

cancer, such as lung cancer or mesothelioma. Lung cancer

is associated with exposure to a variety ofsubstances (e.g',

cigarette smoke) in addition to minerals, but mesotheli-

om4 or cancer of the mesothelium (lining of the abdom-

inal wall), is commonly associated exclusively with ex-posure to fibrous minerals, predominantly the asbestos

minerals. The potential for a specific mineral to induce

these diseases can be evaluated by numerous techniques'each of which provides different information and has dif-

ferent factors that complicate interpretation. As pointed

out by Rall (1988), there are four basic groups of methods

used to determine the carcinogenic potential of a sub-

stance: epidemiological studies, in vitro studies, in vivo

studies, and prediction of biological activity by compar-ison with a similar mineral (structure-activity relation-

ship).Epidemiological studies. In an epidemiological study'

a substance's health risks are evaluated by determiningthe relationships between human exposure to a substance

and the potential health effects. One approach to epide-

miological studies uses cohorts, or groups of individualswhose lifestyles are similar, to monitor the incidence of

disease in response to exposure to a substance. Ideally,

two groups are chosen such that their difference is only

in the exposure to a specific substance; thus one group

serves as a control against the health efects from other

agents. In many studies, however, no explicit control group

is used, but the study group is compared with national

averages. Epidemiological data are commonly reported

using a standardized mortality ratio (SMR) that com-pares the observed death rate from a disease in the studygroup with the rate expected based on the control group-

Another approach to epidemiological studies uses case

studies to determine lifestyle patterns of individuals af-

flicted with a disease. In both approaches it can be diffi-

cult to assess the effect of other harmful substances towhich individuals were exposed, such as other mineral

dusts or tobacco. In other words, an epidemiological studylooks for patterns in the incidence ofa disease and, thus,permits only an indirect determination of the cause of

that disease. An obvious advantage of an epidemiologicalstudy, however, is that it attempts to determine the actual

effect of mineral-dust exposure on humans exposed under

typical conditions.An important aspect of epidemiological studies is the

characteization of exposure, which can be achieved by a

number of techniques. A dust's mineral content, particle

size and shape, and areal distribution can be measured

directly in an environment by collecting air samples or

soil or dust samples. Such measurements are useful forproviding detailed information concerning current ex-posure conditions. However, onset of disease can occur

20-30 yr after exposure to a mineral dust, and currentexposure conditions may differ significantly from previ-

ous exposure conditions. Furthermore, dust characteris-

tics may fluctuate in some environments, causing an in-correct estimation of exposure.

A direct characteizalion of the dusts to which an in-dividual was exposed can be made by analyzing lung tis-sue or expectorated sputum. A variety ofprocesses occurin the lung following exposure to dust (Lehnert, 1990:Schlesinger and Driscoll, 1989), and many of these resultin a clearance of dust particles via the mucociliary esca-lator within the lung and trachea. Thus, sputum providesa means of directly sampling some of the dust particlesbeing cleared from the respiratory system. This processcan continue over a prolonged period after exposure. Fre-quently, these particles become coated with femrginousmaterial believed to be derived from proteins; such coat-ed particles are referred to as ferruginous bodies. particleconcentration in the sputum has been used as an indi-cation of current particle content in the lung (S6bastienet al., 1984). Analysis of sputum samples is compara-tively simple and inexpensive. However, high lung bur-dens-i.e., -10000 femrginous bodies per gram of drylung (S6bastien et al., 1984)-are required before particlesare detected, and exposure estimates are biased to theextent that the sample includes only those particles beingcleared. An accurate assessment of the mineral contentoflungs can be obtained even for lower lung burdens byanalyzing lung tissue obtained during surgery or a post-mortem examination (e.g., Churg et al., 1984). However,biopsy of lung tissue is complicated by the heterogeneousvariation ofparticle deposition and retention at differentsites within the lung, so lung samples are needed fromseveral locations to estimate an average lung burden.

Incidence of disease is typically estimated by severalmethods. Clinical examination of at-risk individualscombined with chest X-rays can often detect early indi-cations ofdisease, but to assess the subjective factors as-sociated with the grading of a chest X-ray, the same setof X-rays must be read by several individuals. Death cer-tificates provide another means of estimating the inci-dence of disease. However, this method can result in in-correct estimates, since the accurate classification of aspecific disease often requires more extensive analysis thanis commonly performed during a postmortem examina-tion. Finally, in some cases, biopsies can be performedon lung tissue from diseased individuals, and lungs andother organs can be removed and examined after an in-dividual from an exposed group has died, as is done withasbestos workers in South Africa, for example. Even so,there is some concern that neoplasias (malignant tumors)can be classified incorrectly, resulting in either an over-estimation or underestimation of the incidence of disease.

In vivo studies. Animal models are used extensively tostudy the effects ofexposure to mineral dusts. Ideally, ananimal species is chosen such that its response to a spe-cific substance closely resembles the response observedin humans under similar conditions. With such an animalmodel, the complete biological effect of a substance canbe studied under various exposure conditions. In prac-tice, however, responses observed in animal models are

GUTHRIE: EFFECTS OF INHALED MINERALS 227

not identical to responses observed in humans, so pre-diction of a human response using results from an in vivoexperiment is not always straightforward. In mineral-dustresearch, rats and mice are the most commonly used an-imal models; however, guinea pigs, sheep, dogs, ham-sters, monkeys, and rabbits have also been used. Pott(1980) reviewed some of the in vivo experiments con-cerning the biological effects of mineral fibers and dis-cussed the differences in response among species.

Because disease must be induced more rapidly in ani-mals than it is induced in humans, in vivo experimentscommonly use exposure methods that differ significantlyfrom exposure conditions experienced by humans. Dis-ease in humans often occurs up to 20-30 yr after initialexposure to a dust; however, most lab animals live lessthan 20-30 yr. Hence, disease is induced more rapidly inan in vivo experiment than would be expected under nat-ural exposure conditions. Typical in vivo experimentsemploy one of three exposure methods: (l) intratrachealinjection of a dust-saline solution into the target organ;(2) direct application ofthe dust to the target organ (e.g.,intrapleural and intraperitoneal instillations); or (3) in-halation in a dust-rich environment (e.g., l-50 mg/m3 or- 500-2500 fibers/ml). (The current regulatory standardfor occupational asbestos exposure in the U.S. is <0.2fibers,/ml; however, 500/o of all asbestos exposure levelsin U.S. schools lie within l0-6-10 3 fibers/ml, accordingto the Environmental Protection Agency, 1986.) The routeof entry for the dusts in such experiments clearly differssubstantially from the typical route of entry in humans(inhalation in a comparatively dust-poor environment).However, even for inhalation experiments conducted withreasonable dust levels, exposure would differ from thatexpected in humans, since the differences between therespiratory systems of laboratory animals and humans(e.g., airway size and shape, breathing patterns, clearancemechanisms) introduce a sampling bias on the particlesreaching the lungs (e.g., Oberdiirster and Lehnert, 1990).Short-term (2-3 yr) animal experiments also inadequate-ly model human exposure in that short-term experimentsdo not consider the long-term (10-30 yr) alteration ofmineral particles (such as dissolution or surface modifi-cation) by the biological medium.

In vivo studies can provide important information, suchas (l) the effect of mineral dusts on a living organismincluding types, incubation periods, and severity of dis-eases, translocation (migration) and clearance rates ofparticles from the site of initial exposure, and cellularresponses to exposure; (2) the effect ofvarious exposureconditions; (3) an evaluation of the risk to humans; (4)the elucidation of pathogenic (disease causing) mecha-nisms; and (5) the identification of potential treatmentmethods. However, such experiments are time consum-ing (up to several years duration), expensive, and difficultto interpret (unless a very strong or very weak effect isfound) (Rall, 1988). Furthermore, the use of data from invivo experiments to predict human response can be com-plicated by a variety of experimental factors, including

228

differences between human and animal response to ex-posure, the degree and method of exposure, and the useof animal strains particularly susceptible or resistant todisease. These two last factors additionally make com-parison of results from different studies difficult unlessidentical experimental procedures were used. When pro-vided in the original report, animal strains will be in-cluded here in the review of in vivo data to allow com-parisons to be made between studies.

In vitro studies. In vitro experiments use specific cellsto determine a mineral's biological activity and are com-monly used because they are rapid and relatively inex-pensive. One of the more commonly used in vitro meth-ods is the Ames test (Ames et al., 1975), which usesmutation rates in bacteria as a measure of carcinogenicpotential. However, the asbestos minerals are one of onlytwo suspected carcinogens (the other being conjugatedestrogen) that do not appear mutagenic in a bacterial as-say (Chamberlain and Tarmy, 1977; Shelby, 1988). Theimplications of this remain incompletely understood.

Eukaryotic mammalian cells are also used for in vitroexperiments to test a mineral's biological activity, withred blood cells (RBCs), macrophages, and epithelial cellsbeing the most commonly used cell types. These cell typesare also found in the lung where they can potentially in-teract with inhaled dusts. Hemolysis experiments test theability of a substance to destroy or lyse RBCs by incu-bating RBCs in contact with the substance and then mea-suring cell viability (the release of hemoglobin is an indexof cell destruction). In vitro experiments with other celllines also test for cytotoxicity (the ability ofa substanceto kill a cell) normally either by (l) the cellular exclusionof a vital dye (where dead cells allow penetration of thedye but living cells do not); (2) the reduction in the abilityofthe cells to develop into colonies; or (3) the release ofenzymes indicative of an increase in cell-membrane per-meability or cell death, where membranes of healthy cellsare impermeable to the enzymes. Heppleston (1984) pre-sented an excellent discussion of information that can bederived from cytotoxicity experiments.

In addition to their use in determining cytotoxicity, invitro experiments can be used to determine the genotox-icity (ability to affect genetic material) or mutagenicity(ability to cause mutations) of a substance. As mentionedabove, the Ames test is one such technique. Other meth-ods involve a quantification of mutation by measuringprocesses involving chromosomes or DNA directly, suchas sister chromatid exchange (SCE) or unscheduled DNAsynthesis. O radicals, such as the superoxide anion, arebelieved by some investigators to participate in both fi-brosis and carcinogenesis (Mossman et al., 1989; Moss-man and Marsh, 1989), and the measurement of the gen-eration ofsuch anions by a catalytic reaction involving amineral surface (Pezerat et al., 1989) is another potentialindicator of a particle's mutagenic activity.

Structure-activity relationship. The simplest and leastexpensive method to determine the potential health haz-ards of a mineral dust may eventually prove to be the


prediction of a mineral's biological activity by compan-son with known structure-activity relationships. How-ever, an accurate knowledge of the mechanisms by whicha mineral is toxic is essential for this method to be effec-tive. Several mechanisms are currently proposed to ex-plain the biological activities of minerals (Table I andFig. l). However, these mechanisms remain too poorlyunderstood to allow an accurate prediction based on amineral's structure. Nevertheless, such predictions aremade, primarily based upon the observed correlation be-tween biological activity and particle shape and size (e.g.,Stanton et al., l98l).

It is interesting to note that many of the proposedmechanisms for mineral-induced disease involve chem-ical reactions (e.g., oxidation/reduction) that are similarto reactions that occur in a geological environment. Ageochemist's approach to the study of such reactions dif-fers greatly from the approach taken by most biologicalscientists. Consequently, geochemists can contributeenormously to the study of mineral-induced diseases. Onerole for the geochemist is the identification and charac-terization of active sites on mineral surfaces (the resultsof which would be of interest to both geologists and bi-ologists). By characterizing the appropriate physical andchemical aspects of each sample, biological activity canbe related to a measurable parameter (e.g., surface area,Lewis-acid/base sites per surface area, Bronsted-acid sitesper surface area) and a model for biological activity canbe developed. Furthermore, a sound mineralogical ap-proach would facilitate the design of biological experi-ments that control most potentially active mineralogicalcharacteristics while allowing the active site of interest tobe studied. For example, numerous experiments have beenconducted to test the biological activity of fibrous am-phiboles. As shown in Figure l, however, amphiboleshave numerous potentially active sites. Hence, the resultsof a study on the toxicity of amphibole cannot be asso-ciated with one particular active site. Conversely, if ex-periments were conducted using two amphiboles that dif-fered only in the composition of the octahedral site, thenthe activity of polyvalent cations in amphiboles (activesite 3 on Fig. l) could be determined. The diversity ofmineral species offers a unique potential for characteriz-ing the activity of numerous mineralogical characteris-tics. In addition to natural samples, synthetic mineralscould be used effectively, e.g., by growing zeolites withidentical framework topologies but with various amountsof tetrahedral Al.

Mineralogical aspects

Mineral names. In principle, usage of nomenclature todescribe a mineral should follow strict guidelines. Forspecies names, these guidelines are widely accepted andhave been developed so that a mineral name providesimportant information concerning both structure andcomposition. For example, the nomenclature for amphi-boles is extremely complex and relies on a knowledge ofthe composition and structure of a given amphibole spec-

TABLE 1. Mechanisms of mineral-induced disease

229

Fig. l. Schematic diagram of the amphibole structure viewedalong the c axis. Numbers indicate possible active sites: (l)A-site cations, (2) protons associated with Al substitution in thetetrahedral sites, (3) polyvalent cations in the octahedral sites,(4) protons associated with underbonded O atoms.

sheets for the asbestos minerals. Both the National Insti-tute for Occupational Safety and Health (NIOSH) and atleast one major supplier of asbestos for biological re-search provide data sheets listing incorrect mineral for-mulae for the asbestos minerals. Such incorrect infor-mation may form the basis of mineral identification orinterpretation of results used in some studies on the healtheffects ofasbestos.

Because of this casual usage of mineral-species nomen-clature, it is difficult to interpret the results of a study interms of mineralogical properties, such as structure andcomposition. The improper use of mineral-species namesis comparable to an incorrect usage of nomenclature forcell type or animal species. Ifa report stated that "rodentswere used in the experiments" or, worse, "rats were usedin the experiments" when in fact the experiments wereconducted on mice, the data would be extremely difficultto interpret. Most reports on the in vivo effects of min-erals are extremely specific in describing animal speciesand cell types, yet they often fail to identify mineral spe-cies correctly. Unfortunately, the advantages of a strictusage of mineral-species nomenclature are not fully ap-preciated by many scientists involved in health-relatedmineral research, including both minerals scientists andnonminerals scientists.

Sarnple purity. Another source ofuncertainty in thesestudies concerns purity of the mineral dust. Detailed de-scription of the mineral content is rarely given, and iden-tification of the minerals generally relies upon that madeby the supplier. Even when obtained from reliable sources,however, the exact mineral content of a dust is suspect.Most suppliers provide mineral samples that containmixtures of minerals, even though one mineral may be


Proposed mineralogicalmechanisms

Proposed biologicalmechanisms

Bronsted-acid sitesproton-donor sites associated

with underbonded O atomsresulting from either cationsubstitutions or brokenbonds at a mineral surface

Lewis-acid/base siteselectron-acceptor/donor sites

associated with polyvalentcations

Alkali-cation siteseasily exchangeable cations,

e.9., amphibole A site or ze-olite cage site

Specif ic surface periodicitiesperiodicities that promote a

specific interaction with aparticular molecule, e,9.,DNA-particle interactions re-ported by Appel et al. (1988)

Oxidative stressresulting from the catalytic pro-

duction of oxygen radicals ata mineral surface

Genetic alterationby a number ol mechanisms, in-

cluding transfection of inter-cellular DNA and DNA-particleinteraction during mitosis

Incomplete phagocytosisresulting in the release of cyto-

toxic enzymes by the cell

Nofe.'There is no implied correlation between opposing mineralogicaland biological mechanisms.

imen (Leake, 1978). Hence, use of the species name rie-beckite defines a mineral as a clinoamphibole (i.e., a dou-ble-chain silicate with a specific stacking order) with acomposition of NarFerSirOrr(OH)r. Varietal names, how-ever, are not so strictly defined. So, although the varietalname "crocidolite" is commonly used to describe asbes-tiform riebeckite, the identification can be based on theblue color and asbestiform habit but not on composition-al or structural information.

Numerous examples illustrate the problems that canresult from inaccurate usage of mineral-species nomen-clature. "Amosite" is a term used to describe brown as-bestos. The term originates from an acronym for the As-bestos Miners of South Africa. Although the term isfrequently used to describe asbestiform cummingtonite-grunerite, it has also been used to describe ores containingmixtures of asbestiform amphiboles. This ambiguity hasled to conflicting definitions of "amosite," i.e., "amosite"has been defined as a varietal name for asbestiform ferro-gedrite by some (e.g., Roberts et al., 1990). Hence, theterm "amosite" provides limited information pertainingto the mineral content of the sample.

In reports on the effects of zeolites, erionite and mor-denite are often referred to as fibrous and nonfibrousequivalents, despite the fact that both are normally fi-brous and have different structures and composition.Furthermore, the identification of one or the other is typ-ically made using qualitative analytical TEM (ATEM)based on the presence or absence ofspecific exchangeablecations. Similar examples of the incorrect usage of min-eral names occur throughout the literature on the healtheffects of dusts. Hence, the reported mineral content ispotentially suspect, unless adequate data were used foridentification.

The lack of interest in the importance of a mineral'sstructure and composition is further demonstrated by data

230 GUTHRIE: EFFECTS OF INHALED MINERALS

the dominant constituent. Mineral impurities, even rnsmall quantities, may have a significant effect on a bio-logical response. For example, recent studies (e.g., Churget al., 1984) show that the lungs ofworkers exposed tochrysotile contain abundant amounts of fibrous tremo-lite, a minor contaminant of chrysotile ores. Churg et al.(1989) found that the rate of mesothelioma is stronglycorrelated with tremolite but much less so with chryso-tile. Continuing with the analogy above, the use of im-pure mineral samples is equivalent to running an in vitroassay with lung cells, which would consist of several typesof cells (e.g., macrophages and epithelial cells). This wouldnever be done in an in vitro experiment, as each cell typeresponds differently.

Mineral identification. Most of the studies reviewedbelow generally characterized samples by light micros-copy or transmission electron microscopy (TEM) and lesscommonly by ATEM. Electron diffraction analysis israrely used to determine mineral content, despite the factthat most of the particles cannot be identified uniquelybased on morphological and compositional data alone.Particle identification by TEM can be time consumingwhen large numbers of particles are included, especiallywhen ATEM and electron diffraction are used. Hence,results are potentially affected by poor counting statisticsand incorrect identification ofthe particles (when electrondiffraction is not used). Minerals present in dust can alsobe determined by quantitative X-ray diffraction (XRD).Davis (1990) measured reference intensity ratios (RIRs)for chrysotile and amphibole asbestoses and demonstrat-ed that the minerals in asbestos mixtures can be deter-mined with a lower detection limit of -0.5-2.0 wto/o.Puledda and Marconi (1990) also reported a low detec-tion limit (2 pg) for chrysotile in various matrices. Chi-pera and Bish (1989) and Bish and Chipera (1991) usedRIRs to determine erionite concentrations in dusts andreported detection limits as low as 100 ppm. Quantitativeanalysis of mineral content using the Rietveld methodwith X-ray difraction data (Snyder and Bish, 1989) mayprove to be even more successful than the use of RIRs,since the Rietveld method effectively addresses problemsassociated with peak overlap and compensates somewhatfor the high degree ofpreferred orientation exhibited byfibrous and platy minerals. However, no biological stud-ies using this approach have been reported to date.

Assessing human risk

As indicated above, many of the results ofexperimentson mineral-induced pathogenesis do not relate directly torisks to humans. For example, though a mineral may behighly active in an in vitro assay, it may pose little riskto humans under normal conditions (e.g., kaolinite). Hu-man response relates to a number of factors, includingthe pathogenicity of the sample, residence time in vivo,dose, and variations in individual response (e.g., physicalcondition of the person, smoking history, propensity to-ward a specific disease). Hence, a mineral such as kaolin-ite may be highly active at the cellular level, but it does

not reside long enough in the lungs to induce disease.Alternatively, a mineral may show a positive response ina given assay, but the mechanism tested by the assay isnot involved in pathogenesis in humans.

Rrvrnw oF DATA

Numerous data are reported that describe the biologi-cal effects of mineral dusts. However, most such studiesprovide only limited mineralogical detail. Hence, the re-sults from these studies may not reflect results one wouldanticipate from a mineralogically pure sample. Studiesthat reported only limited mineralogical data may haveused samples that were (l) mineralogically homogeneous,containing the mineral described; (2) mineralogically het-erogeneous, containing numerous minerals; or (3) min-eralogically homogeneous, containing a mineral differentfrom the one described. In general, it is not possible basedon the published data to determine to which category asample belongs. In the absence of data to the contrary, Ibelieve it should be assumed that the mineral content ofa sample is as described by the original investigators.Nevertheless, this adds uncertainty to any interpretationbased on these data, and this uncertainty should be rec-ognized. In order to clarify these uncertainties, some in-vestigators have supplied me with their samples, and thesesamples are being characterized using quantitative X-raydiffraction analysis (Guthrie and Bish, unpublished data).Where preliminary results are available, these will be giv-en. Those investigators that have supplied samples arecommended for their desire to augment their studies withdetailed mineralogical information.

For those studies that did present mineralogical infor-mation (ranging from a generalized locality to completedescriptions, including compositional analysis), the min-eralogical detail will be included with the summary ofresults. When no mineralogical information was given,only a mineral or material name will be listed.

Related to the poor mineralogical detail is potentiallyinaccurate usage of mineral-species names. The use ofunaccepted mineral names, rock names, and compoundnames is rampant in the biological literature. Some as-sumptions, therefore, were made in order to categorizethese studies with respect to mineralogy. For instance, forthe purpose of categorizing it was assumed that "FerOr"and "iron-ore dust" refer to hematite, "bentonite" refersto montmorillonite, and "attapulgite" refers to palygor-skite; the quoted terms are retained in the review to allowthe reader to recognize assumptions by the author. Fur-thermore, when mineral group terms (such as carbonateor mica) were used, these are also retained in the review.This is not meant to imply that either the quoted termsor the mineral group names are mineral-species names,but rather it serves to categorize the study as appropri-ately as possible based on the probable major miheral.

Oxides and hydroxides

Most studies on oxide- and hydroxide-bearing dustssuggest that some samples can produce fibrosis in vivo

GUTHRIE: EFFECTS OF INHALED MINERALS z 3 |

but are generally relatively inactive minerals. Exceptionsmay include oxides containing Cr (not necessarily min-eral samples) and fibrous brucite. Fibrous brucite may,in fact, be extremely active, with exposure resulting infibrosis, carcinogenesis, or both.

Hematite. Epidemiological studies srrggest that expo-sure to hematite-bearing dust alone under modern min-ing conditions-i.e., low dust exposure, ventilation, wetdrilling-does not increase the risk of lung cancer. How-ever, individuals exposed under unfavorable mining con-ditions-i.e., high exposures to dusts (especially whencontaminated with silica), radon, and tobacco smoke-do show a higher than expected incidence of respiratorydisease.

Lawler et al. (1985) found no overall excess of mortal-ity due to lung cancer (SMR : 0.97 for all cancers andSMR : 0.94 for lung cancer; SMRs based on U.S. whitemales) among 10403 underground miners in Minnesotawho were exposed to iron-ore dust (hematite * "limo-nite," silica, phosphates, and other oxides, as given byLawler et al., 1985). In fact, they found a lower thanexpected mortality for respiratory disease overall (SMR: 0.79). However, they reported no information aboutexposure conditions, i.e., quantitative mineral content ofthe dusts, particle sizes, or mass concentrations. Lawleret al. agreed with suggestions by previous workers (e.9.,Boyd et al.,19701, Radford and Renard, 1984) that ob-served excesses in mortality due to lung cancer in iron-ore miners reflect exposures to other factors, such as ra-don and radon daughter products (RRDs), silica, tobaccosmoke, and diesel fuel, since exposure to these factors wasminimal in their cohort compared with cohorts of iron-ore miners studied previously.

Chen et al. (1990) also suggested that exposure to RRDsand silica could explain excesses of mortality due to lungcancer in hematite miners. They studied 6444 male work-ers associated with hematite mining in China, 5406 ofwhom were involved in underground operations, and re-ported SMRs (based on age-specific death rates for Chi-nese males) for lung cancer ranging from - 1.0 at the 950/oconfidence level for zero-to-medium dust exposure to>2.7 for heavy dust exposures. However, exposure toRRDs is highly correlated with dust exposure; hence, theindividual effects ofthe two could not be separated. In-cidences of other respiratory diseases, such as silicosisand tuberculosis, are also correlated with the incidenceoflung cancer, but no data on silica exposure were given.Cigarette smoking shows a positive relationship with lungcancer as well, as indicated by an SMR for lung cancerin smokers of 2.7-6.3 (950/o confidence interval or CI). Inthe cohort of Chen et al. the use of modern mining con-ditions (wet drilling and ventilation) has lowered airbornedust exposure significantly (from > 100 mglm3 to < 5 mglm3), and workers exposed only under the improved con-ditions may show a lower SMR for lung cancer (l.I-4.6,950/o CD than those first exposed prior to the use of wetdrilling and ventilation (2.9-7 .4,950/o CI).

In vivo experiments indicate that hematite-bearing

samples are biologically inactive. Pott and coworkers (Pottand Friedrichs,1972:' Pott et al.,1974) found no fibrosisor tumors in 80 Wistar rats 530 d after intraperitonealinjections of hematite (source not given), whereas in thesame study, Wistar rats showed fibrosis and up to a 40o/oincidence of tumors when injected with chrysotile and a550/o incidence of tumors following the injection of silicaglass. Vorwald and Karr (1938) found that hematite dustinduces no tumors in guinea pigs or rats following inha-lation of the dust; no information was given concerningthe source ofdusts or exposure levels. However, asbestos(type not given) also failed to induce tumors in guineapigs in the same experiment. Finally, Mossman andCraighead (1982) found that hematite (IIT Research In-stitute, Chicago) does not induce tumors in golden Syrianhamsters following subcutaneal implantation of in vitro-exposed tracheas; however, hematite is nearly as effectivea cocarcinogen as riebeckite asbestos [Union Internatio-nale Contre le Cancer (UICC) standardl when pretreatedwith a polycyclic aromatic hydrocarbon.

In vitro experiments suggest that hematite-bearingsamples are noncytotoxic and nongenotoxic. Dubes andMack (1988) used an in vitro technique to test the abilityof a variety of materials to mediate the transfection ofmammalian cell cultures; transfection of cells is the pro-cess of introducing foreign genetic material into a cell andis one proposed mechanism for the carcinogenicity ofmineral dusts (e.g., Appel et al., 1988). Their data showthat FerO, (reagent grade from Matheson, Coleman, andBell Company) is only slightly effective as a mediator.For comparison, asbestos (from a variety ofsources) andCrrO, (from Matheson, Coleman, and Bell) were -3-7

times more effective mediators. Witmer and Cooper(1983) also reported that Fe'O, is nonmutagenic whereasCrrO, is mutagenic, as determined in a modified Amestest.

Boehrnite, goethite, and lepidocrocite. In vivo experi-ments suggest that samples containing iron and alumi-num hydroxides may be slightly active in the lung. In-halation experiments by Gardner et al. (1944) showed noeffect of boehmite laths [measuring -7.5 x 30.0 nm;mineral identification confirmed by King et al. (1955)using TEM and XRDI on the lungs of guinea pigs. How-ever, using the same material, King et al. (1955) observedthat severe and rapid pulmonary fibrosis develops in ratsafter a direct injection of boehmite-saline solution intothe lungs. Stacy et al. (1959) extended the study by Kinget al. (1955) to include an additional sample of boehmite(better crystallized and having a mean size of -3 pm)and samples of goethite and "lepidocracite" (presumablylepidocrocite) measuring <0.5 pm and -0.5 x 2 p,fi,respectively. They demonstrated that the boehmite sam-ple used in the experiments by Gardner et al. (1944) pro-duces a dose-dependent fibrogenic response, whereas thelarger-grained, better-cryst allized boehmite is much lessfibrogenic. Goethite- and lepidocrocite-bearing samples,however, are only slightly fibrogenic, even at higher dosesthan boehmite. Inhalation experiments by Campbell


(1940) using "the precipitated brown oxide of iron (FerOr,HrO), British Drug Houses" produced a22.7o/o incidenceof lung tumors in mice, compared with 6.80/o in the con-trol group and 17.60/o in a group exposed to "precipitatedsilica" dust; tumors did not develop until after 300 d,with most developing 600-900 d after first exposure. An-imals were exposed to 0.5 g of dust per hour, 6 h per day,5 d per week for I yr.

Brucite. In vivo experiments suggest that samples con-taining fibrous brucite (described as "nemalite" in moststudies) are both fibrogenic and carcinogenic. Pott andcoworkers (Pott and Friedrichs, 1972; Pott et al., 1974)found that fibrous brucite, when intrapleurally injectedin Wistar rats, produces fibrosis comparable to that pro-duced by silica or chrysotile injections and exhibits a tu-mor rate (62.50/o) exceeding those of silica (550/o) andchrysotile (400/o); palygorskite, however, exhibits a 650/otumor rate in the same test. Wagner et al. (1973) foundthat 20 mg of a sample containing fibrous brucite admin-istered to Fischer 344 rats by intrapleural injection in-duces mesotheliomas at a rate of 560/0. Their sample wasobtained from a Canadian mine and contained chrysotile,though no estimate for the amount of chrysotile contam-ination was given. It should also be noted that chrysotilefrom this same mine induces mesotheliomas under thesame conditions at a slightly higher rate (610/o).

In vitro experiments further indicate that samples con-taining fibrous brucite are cytotoxic to a variety of cellIines. Chamberlain and Brown (1978) showed that fi-brous brucite (same material as used by Wagner et al.1973) reduces the colony-forming efficiency of Chinese-hamster lung cells at doses roughly equivalent to thosefor a comparable cytotoxic response in experiments witheither amphibole or serpentine asbestos. The dose re-quired to reduce cloning efficiency to 500/o is 12 pg/mLfor fibrous brucite compared with 9 pg/mL for riebeckiteasbestos and 17-26 p{mL for chrysotile; talc is nontoxicat 50 pglmL, the highest dose used. Jaurand et al. (1980)found that fibrous brucite is cytotoxic to human RBCsand rabbit alveolar macrophages. They demonstrated thatboth the hemolltic and cytotoxic activities of fibrousbrucite are intermediate to those of chrysotile or riebeck-ite asbestos. Finally, Pezerat et al. (1989) found that fi-brous brucite is comparable to chrysotile and much moreeffective than amphibole asbestos in its ability to catalyzethe production of O radicals, a step of potential impor-tance in both fibrogenesis and carcinogenesis.

The 1:l layer silicates and chlorite

Most studies of samples containing l: I layer silicatesor chlorite suggest that some samples can produce fibrosisor tumors in vivo and can be highly active in vitro. How-ever, epidemiological data on exposure to kaolinite-bear-ing dusts suggest that fibrosis is induced only in extraor-dinary conditions, i.e., high exposures or in the presenceof other pulmonary complications. Although these min-erals may be cleared rapidly from the lung (and hence arenot pathogenic in humans), their in vivo and in vitro

activities may provide clues to the mechanisms of min-eral-induced pathogenesis.

Kaolinite and halloysite. Epidemiological studies sug-gest that kaolinite-bearing dust is fibrogenic only underextraordinary conditions, i.e., high dust conditions or ex-posure combined with another respiratory disease, suchas tuberculosis. Hale et al. (1956) reported case studiesof seven kaolinite workers (primarily baggers exposed toextremely high dust conditions) who showed indicationsof respiratory disease, as determined by clinical exami-nations including chest X-rays. Autopsies of two of themen revealed fibrosis associated with large amounts ofkaolinite, mica, and amorphous silica. One of the autop-sies, however, also noted tuberculosis; dead tuberculosisbacilli enhance the fibrogenic effect ofkaolin dust in an-imals (Kettle, 1934; Attygalle et al., 1954). Similar ob-servations were reported in a case study by Lynch andMclver (1954). In a cohort study, Sheers (1964) foundfibrosis in up to l3olo of kaolinite workers exposed to highdust levels. Tuberculosis was uncommon in his cohort.However, fibrosis was highly correlated with high dustexposures and length ofemployment. More recent studiesconfirm that exposure to high dust levels (particularlysilica-bearing dusts) during kaolin mining can be corre-lated with abnormalities in chest X-rays (e.g., Kennedyet al., 1983; Oldham, 1983; Sepulveda et al., 1983; Ogleet al., 1989). However, some of these abnormalities maynot reflect the onset of fibrosis (Oldham, 1983). Lapenaset al. (1984) confirmed the presence of kaolinite in pul-monary tissue from five kaolin workers with pneumo-coniosis; silica was not present in the lung samples.

In vivo experiments reported thus far on the fibrogenicpotential of kaolinite-bearing dusts are inconclusive. Ket-tle (1934) observed no fibrosis in guinea pigs followingintratracheal injection of kaolinite (British Drug Houses;the sample contained quartz and "very numerous" seri-cite fibers), though, as indicated above, he did find thatexposure to kaolinite and dead tuberculosis bacilli doesresult in fibrosis. King and Harrison (1948) used directinjection into the lung to study the effects of two kaolinitesamples on rats. Unfortunately, one of the experimentsused kaolinite samples containing 35.68 wto/o carbonateminerals (species not given), whereas in the other exper-iment, which used a comparatively pure sample of ka-olinite, only two rats survived more than l0 d after ex-posure. Neither of these rats developed fibrosis. Mossmanand Craighead (1982) found that kaolinite (3-5 pm indiameter; Georgia Kaolin Company) does not induce tu-mors in golden Syrian hamsters following subcutanealimplantation of in vitro-exposed tracheas and is a slight-ly less effective cocarcinogen than UICC crocidolite whenpretreated with a polycyclic aromatic hydrocarbon. In-halation experiments by Wagner (1990) produced no lungtumors in 20 rats (probably from the Wistar strain) ex-posed over a period of 3-24 months, but a slight fibro-genic response was observed. His samples contained 85-950/o kaolinite, with the remainder consisting of mica,feldspar, and quartz. For comparison, a "nonfibrous ze-

GUTHRIE: EFFECTS OF INHALED MINERALS Z J J

olite" and a "long attapulgite" produced more severe fi-brogenic responses. Wastiaux and Daniel (1990) also usedinhalation-exposure methods to assess the fibrogenicityof kaolin. They reported that their kaolin sample (Cor-nish kaolin dust) induces a moderate fibrogenic responsein Wistar rats. Long-term experiments currently in prog-ress by Maltoni and coworkers (Maltoni et al., 1982; Mal-toni and Minardi, 1989) may provide additional infor-mation on the in vivo activity of kaolinite.

In vivo studies using halloysite-bearing samples, how-ever, suggest that this kaolin-group mineral may be car-cinogenic. Stanton et al. (1981) found that two samplesof halloysite (obtained from the water supply of HongKong) induce a tumor rate of -20o/o in Osborne-Mendelrats exposed by direct application ofthe dust to the pleu-ral surface. For comparison, in the same experiments,amphibole asbestoses induce tumors at rates ranging from0 to 1000/0. Wagner (1982) also reported in vivo data onhalloysite. He observed no mesotheliomas in 40 Fischer344 rats treated by intrapleural inoculation, whereaschrysotile (UICC standard B; derived from Canadian de-posits) induces 22.5o/o mesotheliomas by the same tech-nique. Whether the observed difference in the pathoge-nicities of kaolinite and halloysite is related to particlemorphology or other mineralogical properties (e.g., sur-face characteristics) is not known.

In vitro experiments show that kaolinite-bearing sam-ples are cytotoxic to most cell types studied, though somematerials are noncytotoxic. Low et al. (1980) found thatkaolinite is cytotoxic to rabbit alveolar macrophages; theirsample was a99o/o pure kaolinite (as determined by XRDand energy-dispersive spectrometry) obtained from theGeorgia Kaolin Company. Davies (1983) showed that ka-olinite is also cytotoxic to mouse peritoneal macrophagesbut that treatment of the dust with poly(2-vinylpyridineN oxide) (PVPNO), a class of polymers that inhibit thecytotoxicity of quartz (Holt et al., 1970), almost com-pletely eliminates kaolinite's cytotoxicity. Davies notedthat his sample contained 2o/o mica, as determined byXRD. Dubes and Mack (1988) found that kaolinite (J. T.Baker Chemical Company) is -4-5 times more effectivethan asbestos in mediating transfection of mammaliancell cultures. Gormley and Addison (1983) also foundthat the kaolinite standards of the CMS Clay MineralRepository, KGa-l and KGa-2, are cytotoxic to a macro-phagelike mouse cell line only at high doses. In contrastto the above studies, however, Marks and Nagelschmidt(1959) found that kaolinite is much less cytotoxic to guin-ea pig peritoneal macrophages than silica minerals.Woodworth et al. (1982) used the release of s'Cr to mon-itor changes in cell-membrane permeability and cell deathin Syrian hamster tracheal epithelial cells. They foundthat kaolinite (Georgia Kaolin Company) will cause therelease of s'Cr. Kaolinite is less effective than chrysotileand montmorillonite but more effective than silica.

There is some indication that kaolinite's cytotoxicity isin part related to broken Si-O bonds at the crystalliteedges. As noted above, Davies (1983) found that the

treatment of kaolinite with PVPNO reduces kaolinite'scytotoxicity at amounts less than the total amount ofpglymer that can be adsorbed, implying that only someof the polymer-binding sites may be related to kaolinite'scytotoxic activity. Furthermore, PVPNO is effective atinhibiting the cytotoxic activity of quartz (Holt et al.,1970), suggesting that the mechanisms by which quartzand kaolinite exert their cytotoxic effects are related. Steeland Anderson (1972\ found that the addition of a bac-terium, Staphylococcus aureus, to a kaolinite-NaCl solu-tion at low NaCl concentrations Qa mM) inhibits floc-culation, possibly because of an interaction between thebacterium and the kaolinite crystal edges. Others (Ken-nedy et a1., 1989; Ghio et al., 1990) have shown thatkaolinite (noncalcined Georgian sample) and other pneu-moconiosis-causing minerals function as Fenton catalysts(electron transfer by Fe2* + fsr+ * e ), possibly as aresult ofFe3* adsorbed on its surface. Their studies illus-trate that biochemical mechanisms can be probed effec-tively if mineral samples are selected carefully.

Serpentine, berthierine, and chlorite. Chrysotile is theserpentine mineral that has been studied in greatest detailas a potential health hazard. As indicated above, becauseof the enormous body of literature on the health effectsof chrysotile, this mineral is not addressed directly in thispaper. The interested reader, however, is directed to therecent article by Mossman et al. (1990) and papers byRoss (1981, 1984) for reviews of the research on chrys-otile.

With respect to other serpentine minerals, Woodworthet al. ( I 983) found that antigorite (Ward's Scientific; sam-ple from Arizona) does not induce metaplasia (prolifer-ation of cells) in tracheal mucosa of the golden Syrianhamster in vitro, whereas riebeckite asbestos does; thetracheal mucosa, or lining of the trachea, is a part of therespiratory tract with which inhaled dusts interact. Usingthe same material, Mossman and Sesko (1990) found thatantigorite does not cause the release of 5tCr from hamstertracheal epithelial cells, whereas riebeckite asbestos andchrysotile do. Hence, if antigorite is cytotoxic, it is muchless active than chrysotile.

A berthierine-rich iron orc (40o/o berthierine; samplefrom Lorraine, France) and two Fe-rich chlorites (Pyr6-n6es and Anjou) were studied by Costa et al. (1990), usinga chemical assay to measure the production of activatedO species. Production of activated O species is a mech-anism by which a material can induce a toxic response.They found that the samples were highly active, and theyassociated this activity with the high Fe content. Fe con-tents (reported as FeO) were in the range 12.5-30.0o/a forthe three samples, but they did not show that the Fe wasdirectly responsible for the observed activity. For com-parison, they found that kaohnite (St. Austelle) and quartz(DQ l2) are inactive in the assay. Despite the poor qual-ity of the specimens used, their study is a good exampleof the type of mineralogical-based research that can ben-efit the field of mineral-induced pathogenesis. The bio-chemical mechanisms they investigated with their assay


involved an electron-transfer process at the mineral sur-face, a process similar to many geochemical processes.

The 2:1 layer silicates

Most studies of samples containing 2:l layer silicatessuggest that some samples can produce fibrosis in vivoand can be highly active in vitro. However, epidemiolog-ical data suggest that fibrosis may not be a problem inmodern mining conditions. As with the l: I layer silicates,though 2:l layer silicates may be cleared rapidly from thelung (and, hence, are not pathogenic in humans), theiractivity may provide clues to the mechanisms of mineral-induced pathogenesis.

Talc. In general, epidemiological studies suggest thatexposure to talc-bearing dusts elicits a dose-dependent(albeit minor) response. Kleinfeld eI al. (1967, 1974)studied the mortality in a group of 220 talc miners em-ployed for at least 15 yr between 1940 and 1965. Dustexposures in this group were very high before 1945 (10,-lOs particles/ml) but dropped substantially after 1945(-10'-103 particles/ml); furthermore, miners were ex-posed to a variety of dusts, including talc, serpentine,tremolite, carbonates, and silica. During 1945-1959,Ihemortality rate due to lung cancer was 3.4 times the rateexpected based on U.S. white males in 1957, but the ratedropped to near normal during 1960-1969, possibly be-cause of lower exposure levels. However, because of theexposure to dusts other than talc (particularly tremolite),it is not possible to assign this effect to talc exposurealone. Other studies (Selevan et al., 1979i Brown andWagoner, 1980; Leophonte and Didier, 1990) of talcminers and millers in New England reported similar ob-servations, but one study (Stille and Tabershaw, 1982)found no increases in mortality from lung cancers amongworkers at one New York mine who had no prior workexposure.

Coexposure to amphiboles is likely in many studies oftalc-exposed workers. Cullinan and McDonald (1990)separated seven studies of talc workers on the basis ofsuspected amphibole exposure. Among the three studieswith no suspected amphibole exposure, no mesothelio-mas were reported out of a total of 2540 workers, thougha slight increase in other respiratory malignancies wasobserved in two studies (Cullinan and McDonald, 1990).

In vivo experiments on talc-bearing dusts suggest thattalc is nonfibrogenic and noncarcinogenic. Pott andFriedrichs (1972) observed no fibrosis or abdominal tu-mors following intraperitoneal injection of talc in Wistarrats. In a later experiment using the same technique, how-ever, Pott et al. (197 4) observed a slight incidence of tu-mors (2.50/o) with a latency period (587 d) twice that ob-served for chrysotile or fibrous brucite. Wehner (1980)observed no significant changes in golden Syrian ham-sters exposed to talc baby powder (presumably obtainedfrom the funding agency, Johnson and Johnson); ham-sters exposed to asbestos cement (mineral content notdescribed) exhibited a response similar to the talc re-sponse at comparable exposures. Wehner used asbestos

dust (mineral content not detailed) as a positive control,but comparison is hindered because the control experi-ments used exposures 8 times those used in the talc ex-periments. Stanton et al. (1981) observed a statisticallyinsignificant tumor rate in Osborne-Mendel rats exposedby direct application oftalc to the pleural surface. Wagneret al. (1979) and Wagner (1990) found no lung tumorsamong 96 Wistar rats exposed to talc by inhalation for3- 12 months. Their sample contained 8o/o impurities in-cluding silica, chlorite, and carbonate. Endo-Capron etal. (1990) induced no pleural tumors after intrapleuralinjection of 20-mg talc (Luzenac, France).

In vitro experiments are inconclusive regarding the cy-totoxic activity of talc-bearing dusts. Chamberlain andBrown (1978) found that Italian talc (commercial cos-metic grade; source not given) is noncytotoxic to Chinese-hamster lung cells at concentrations up to 50 pE/mL.However, using the same assay, Pigott and Pinto (1983)reported that talc (source not given) is slightly c)'totoxicat the same concentration; for comparison, Pigott andPinto found that riebeckite asbestos is highly cytotoxicand calcium carbonate is noncytotoxic. Talc is much lesshemolytic than kaolinite or montmorillonite (Wood-worth et al., 19821, Brown et al., 1980). Despite its weakcytotoxicity, talc is an effective mediator in transfection.Dubes and Mack (1988) showed that talc (talcum pow-der, Mallinckrodt Chemical Works) is -2 times moreeffective than asbestos but -2 times less effective thankaolinite in mediating transfection of mammalian cellcultures. Woodworth et al. (1982) found that talc (CyprusIndustrial Minerals Company, I-os Angeles) will affect cellmembrane (in Syrian-hamster tracheal epithelial cells) asmonitored by the release of 5rCr. Talc is approximatelyas active as kaolinite in this assay. However, Endo-Cap-ron et al. (1990) found that talc (Luzenac, France) pro-duces no SCEs in rat pleural mesothelial cells.

Phlogopite, muscovite, illite, smectite, and vermiculite.Only a few epidemiological studies of respiratory diseaseresulting from exposure to dusts containing micas or mica-like clays have been published, and some ofthese suggestthat such samples can elicit a mild, dose-dependent fi-brogenic response at high exposure levels (e.9., Vestal etal., 1943). Exposure to mica-like minerals is generallyaccompanied by an exposure to other minerals (e.g., silicaand amphiboles), and the response to these mineralscomplicates the interpretation of the data (e.g., Heimannet al., 1953; McDonald et al., 1988). For example, somecases of vermiculite-related mesothelioma may be cor-related with amphibole contamination (see Cullinan andMcDonald, 1990, for a review of the studies).

In vivo experiments suggest that samples containingmicas or mica-like clays are slightly fibrogenic. King etal. (1947) found injection of 50 mg of illite dust (sepa-rated from shales in southern Wales) into the lungs ofrats produces no fibrosis unless the clay is pretreated inan HCI solution. Policard (1934) used exposure by in-halation to study the short-term effects (3-30 d) ofgroundwhite mica (from Madagascar; light microscopy showed


the dust to contain both fibrous and polyhedral particles)on the lungs of rats; ground white mica induces a cellularresponse similar to that observed with quartz. Pott et al.(1974) found that biotite is inactive following intraperi-toneal injection in Wistar rats. Sykes et al. (1982) usedintratracheal instillation to study the short-term (<7 d)and medium-term (< 100 d) effects of "bentonite" on Al-derley-Park-derived rats (strain l; specific pathogen free).Though these results show that "bentonite" induces agreater pulmonary response than quartz in the short term,medium-term effects indicate that "bentonite" induces aresponse similar to a saline control. Rosmanith et al.(1990) studied the relationship between surface area andactivity using intratracheal installation of a well-charac-teized muscovite sample in SPF-Wistar rats (Rosmanithet al., 1990; Schyma, 1990). They found that the finestmaterial elicits the greatest fibrogenic response. Brambil-la et al. (1979) reported mild pulmonary lesions in zooanimals exposed to mica dusts. Mineralogical analysis oflung contents indicated the presence of muscovite andillite.

In vitro experiments suggest that samples containingmicas and mica-like clays may be slightly cytotoxic.though some studies suggest that phlogopite and mont-morillonite may be highly c1'totoxic. Pigott and Pinto(1983) studied the cytotoxicity of phlogopite, "hydro-phlogopite" (?), and biotite (distinction not explained) andmuscovite using Chinese-hamster lung cells. All four mi-cas are slightly cytotoxic, with muscovite showing thegreatest effect and being comparable to talc in activity.As mentioned above, riebeckite asbestos is highly cyto-toxic in the same study.

Gormley and Addison (1983) found that samples SAz-land STx-l (calcium montmorillonites), SWy-l (sodiummontmorillonite), and SHCa-l (hectorite) from the CMSClay Repository exhibit a range in toxicities, with SHCa-lbeing slightly cytotoxic (roughly comparable to kaolinitesamples KGa-l and KGA-2) and STx-l being highly cy-totoxic (more qtotoxic than their positive control, quarz)-It should be noted, however, that they reported l0o/o cris-tobalite in STx-1, as determined by XRD, and cristobal-ite is even more cytotoxic than quartz (Marks and Na-gelschmidt, 1959).

Adamis and Tim6r (1978) used peritoneal macro-phages from Sprague-Dawley rats to show that both quartzand "bentonite" (Istenmezeje, Hungary; obtained fromZ. Juhdsz) are cytotoxic, but their modes of action aredifferent. Although quartz alters the permeability of cellmembranes to the enzyme lactate dehydrogenase (LDH),"bentonite" does not. However, "bentonite" does signif-icantly lower the intracellular activity of LDH.

Costa et al. (1990) used a chemical assay to determinethe role ofFe2* in the production ofactivated O species.As found for Fe-rich chlorite and berthierine, Fe-rich bi-otite (Raz6s) is an effective catalyst in this assay, whereasan Fe-poor montmorillonite (Maroc) is an ineffective cat-alyst. In light of the purity of other samples used in thestudy (i.e., iron ore to test berthierine and granite to test

biotite and muscovite), the mineralogical purity of thesespecimens may be of some concern.

Woodworth et al. (1982) found that montmorillonite(American Colloid Company, Skokie, Illinois) will affectcell membrane (in Syrian-hamster tracheal epithelial cells)as monitored by the release of 5'Cr. Montmorillonite isroughly as active as chrysotile in this assay. However,Dubes and Mack (1988) found that "bentonite" (ob-tained from Fisher Scientific Company) is approximatelyone-tenth as effective as asbestos at mediating transfec-tion of mammalian cell cultures.

In contrast, Holopainen et al. (1990) found that phlog-opite (phl8, ann,.) is almost twice as hemolytic as quartzand as cytotoxic as quartz to rat alveolar macrophages(as determined by the release of LDH). After treatmentwith nitric and sulfuric acids, the phlogopite is more he-molytic but less cytotoxic. In their assay, the hemolyticand cytotoxic activities of muscovite are comparable tothose of rutile (a negative control).

Modulated 2:l layer silicates

Most studies on samples containing modulated 2:l lay-er silicates suggest that some samples can produce fibrosisor tumors in vivo and can be highly active in vitro. How-ever, epidemiological data suggest these minerals are atmost mildly active in humans.

Sepiolite. One epidemiological study suggests that ex-posure to sepiolite-bearing dust does not increase the riskof pulmonary disease. Baris et al. (1980) studied 63 se-piolite workers in Turkey involved in trimming, cleaning,and polishing sepiolitic stones. Ten of the 63 showed signsof pulmonary fibrosis, but no relationship was establishedbetween exposure to sepiolite and fibrosis. Sputum wasanalyzed from one of the ten, but no fem.rginous bodieswere observed.

In vivo experiments by Wagner (1982) using Fischer344 rats exposed for I yr through inhalation showed thatsepiolite (termed by Wagner as "European sepiolite,"possessing a fibrous morphology) is as fibrogenic as rie-beckite asbestos. However, sepiolite induces no mesothe-liomas in Fischer 344 rats exposed by intrapleural inoc-ulation, whereas chrysotile (UICC standard B) inducesmesotheliomas at a rate of 22.5o/o. Pott et al. ( I 990) foundthat the response elicited by sepiolite is highly sampledependent. The two sepiolite samples studied by Pott etal. (1990) showed tumor rates of 60lo (Finland) and 670/o(Uicaluaro) following intrapleural injection in femaleWistar rats. Preliminary results of a powder X-ray dif-fraction study indicate that these samples contain signif-icant amounts of other minerals (Guthrie and Bish, un-published data); quantitative mineral content data are notavailable yet, so it is not possible to correlate purity withbiological activity.

In vitro experiments by Hansen, Mossman, and co-workers (Hansen and Mossman, 1987; Mossman et al.,1989) indicate that sepiolite (Minerals Research) is ca-pable ofinducing the release ofthe superoxide anion fromboth hamster and rat alveolar macrophages in a dose-


dependent manner. In hamster alveolar macrophages, therelease induced by sepiolite is comparable to the releaseinduced by erionite and riebeckite asbestos; however, inrat alveolar macrophages, sepiolite is less active than er-ionite or riebeckite asbestos in eliciting a response.Chamberlain et al. (1982) found that long-fiber sepiolite(source not given) is as cytotoxic as riebeckite asbestos(UICC standard) to mouse peritoneal macrophages (asdetermined by release of LDH) and human type II alve-olar cells (as determined by the formation of giant cells),but less cytotoxic than riebeckite asbestos to Chinese-hamster lung cells (as determined by reduction in cloningefficiency); short-fiber sepiolite (source not given), how-ever, was determined to be noncytotoxic in the same ex-penments.

Palygorskite ("affapulgite"). Epidemiological data sug-gest that exposure to palygorskite-bearing dusts may in-crease the risk of lung cancer among whites (Waxweileret al., 1988). Waxweiler et al. studied a cohort of 2302miners and millers from an "attapulgite" company in theUnited States. They reported SMRs of <1.0 (based onU.S. males) for nonmalignant respiratory disease in allraces (0.23-0 .7 6, 90o/o CI) and race-specific SMRs for lungcancer (1.21-2.93 in whites, 900/o CI: 0.21-1.12 in non-whites, 900/o CI). Respirable dust exposures were < 5 mg,/m3, but no information concerning mineral content wasgiven except to note that the only fibrous mineral ob-served is "attapulgite clay." No mineral content is re-ported for the dust to which their cohort was exposed.Instead, reference was made to the "typical" mineral con-tent of "attapulgite" clay mined in the United States asreported by Haden and Schwint (1967); a "typical" dustwould thus consist of "70-800/o attapulgite; l-150/o mont-morillonite, sepiolite, and other clays; 4-80lo quartz; andl-50/o calcite or dolomite" (Waxweiler et al., 1988). Sorset al. (1979) reported a case study of a mining engineerwho exhibited signs of respiratory disease following a 2-yrexposure to "attapulgite." Lung lavage fluids suggestedheavy particle burdens; XRD gave a pattern "similar tothose of mineral attapulgite."

In vivo experiments have suggested that palygorskite-bearing dusts are mildly active in the lung, though somesamples can be very active. Stanton et al. (1981) showedthat "attapulgite" is slightly tumorigenic in Osborne-Mendel rats following direct application of the dust to thelungs. Experiments using two different samples resultedin tumor rates of 8 + 5.3o/o and I I + 7.5o/o, samples werefrom Attapulgus, Georgia, and >900/o pure, the remain-der consisting ofquartz. Jaurand et al. (1987) found that"attapulgite" (French; obtained from a deposit in Mormoi-ron) is nontumorigenic following intrapleural injection inspecific pathogen-free Sprague-Dawley rats, whereas inthe same experiments, chrysotile induces tumors at a rateof l9-52o/o, depending on particle size. However, Wagner(1982) observed mesothelioma rates of 12.5-25o/o (de-pending on specimen preparation method) for "attapul-gite" (Spanish) following intrapleural inoculation of Fi-scher 344 rats; chrysotile (UICC standard B) exhibits a

comparable mesotheliom a rare (22. 5o/o). In his inhalationexperiments, Wagner (1982) showed that "attapulgite"(Spanish) is as fibrogenic as riebeckite asbestos, but hereported negative results in the two experiments for an-other "attapulgite" (American). B6gin et al. (1987, 1990)used a bronchoalveolar lavage technique to monitor thecellular response in lungs of sheep exposed to "attapul-gite" (from northern Florida). Exposure results in increas-es in cell numbers and enzyme levels, comparable to thoseobserved after similar experiments using the UICC as-bestos standards. No fibrosis was observed at the end ofthe study, but the elevated levels of enzymes indicate thatthe "attapulgite" is cytotoxic in vivo. Cofrn et al. (1989a)reported a l.4o/o incidence of mesotheliomas in rats in-jected intrapleurally with "attapulgite" from Georyia andFlorida compared with a l.3olo incidence in the controlgroup. Pott et al. (1974, 1990) found that "attapulgite"is carcinogenic at rates from 3.5-400/o following intra-pleural injection in Wistar rats, i.e., the response is sam-ple dependent. Preliminary results of a powder X-ray dif-fraction study indicate that these samples containsignificant amounts of other minerals (Guthrie and Bish,unpublished data).

In vitro experiments have indicated that palygorskiteis as hemolytic as chrysotile, but in other nonerythrocytecell types palygorskite is at most slightly cytotoxic and isnongenotoxic. Bignon et al. (1980) showed that "attapul-gite" (Spanish) was -8 times more hemolytic to humanred blood cells than chrysotile (UICC standard A; derivedfrom Rhodesian deposits). Perderiset et al. (1989) foundthat "attapulgite" (Senegalese; obtained from Rh6nePoulenc) is hemolytic but that pretreatment of the dustwith lipids or proteins (material similar to cell mem-branes or extracellular lung fluid) reduces the hemolyticactivity. Nadeau et al. (1983) reported that "attapulgite"is as hemolytic as chrysotile (UICC standard B) and morehemolytic than sepiolite or erionite.

In contrast, in vitro experiments using cells other thanRBCs have suggested that palygorskite-bearing dusts aregenerally inactive, although the activity varies greatly asa function of the surface characteristics of the sample.Woodworth et al. (1983) found that palygorskite (CMSClay Repository sample from Nevada) might have a slighteffect on cultured hamster trachea, but the effect is notstatistically different from the control group; riebeckiteasbestos and fiber glass, however, test positive statisti-cally in the same assay. Jaurand et al. (1987) found that"attapulgite" (French) may be cytotoxic to rat pleuralmesothelial cells only at high doses, whereas chrysotile isgenerally much more cytotoxic. Reiss et al. (1980) dem-onstrated that palygorskite (Attapulgus, Georyia) is muchless cytotoxic than "amosite" asbestos to human-embry-onic, intestine-derived epithelial cells. Pezerat et al. (1989)found that "attapulgite" (Senegalese) is inactive in cata-lyzing the production of O radicals, and Achard et al.(1987) found that the same material does not induce SCEsin rat pleural mesothelial cells. Renier et al. (1990) foundno unscheduled DNA repair synthesis in rat pleural me-

GUTHRIE: EFFECTS OF INHALED MINERALS z J t

sothelial cells following treatment with "attapulgite"(Mormoiron region, France). Chamberlain et al. (1982)found that long-fiber "attapulgite" (source not given) ismore cytotoxic than riebeckite asbestos (UICC standard)to mouse peritoneal macrophages (see description of as-says in the section on sepiolite) but less cytotoxic thanriebeckite asbestos to Chinese-hamster lung cells; short-fiber "attapulgite" (source not given), however, is slightlycytotoxic to mouse peritoneal macrophages and noncy-totoxic to Chinese-hamster lung cells. Nolan et al. (1991)further demonstrated that the in vitro activity of paly-gorskite varies between samples by showing that amongnine palygorskites that possess different surface charac-teristics there is a corresponding range in hemolytic ac-tivity.

Zeolites

The biological activity oferionite has been studied ex-tensively, and all data indicate that it is extremely activein humans, in vivo, and in vitro. Data on other zeolitesare less conclusive, particularly in light ofthe poor qual-ity of the samples studied.

Erionite. Epidemiological data suggest that exposure toerionite-bearing dusts increases the risk of mesotheliomain humans, even at much lower exposure levels than re-quired for amphibole asbestos-induced mesothelioma.Earlier epidemiological studies in the Cappadocian re-gion ofTurkey revealed outbreaks ofasbestos-related re-spiratory diseases, including mesothelioma (Baris et al.,1979). Initially, it was assumed that asbestos present inthe stucco used in that region was the cause ofthese dis-eases; however, Baris et al. (1979) found outbreaks invillages in which the stucco does not contain asbestos.Later studies focused on exposure to erionite as the cause.Baris et al. (1987) found zeolite fibers (as determined byATEM) in air samples from affected villages, although airsamples from some of these villages additionally containfibrous tremolite. Lung contents as determined from spu-tum samples (S6bastien et al., 1981, 1984; identificationby ATEM and electron diffraction) and biopsies (Baris etal., 1987;' identification by ATEM) also indicate that in-dividuals from these regions have been exposed to botherionite and asbestos (chrysotile, tremolite asbestos, andriebeckite asbestos), though the amount of zeolite ex-ceeded the combined amounts of asbestos (on a per-fiberbasis) in the two samples of human lung contents.

Mumpton (1979) investigated the mineral content ofdusts in the Cappadocian region. He found that erionitewas present in samples from two villages in which meso-thelioma rates are high, although erionite is abundantonly in one of those villages. He also found that erioniteis abundant in a third village, Sarihidir, which at thattime had no reported cases of mesothelioma. Mumptonconcluded, therefore, that the geographic distribution oferionite is inconsistent with the distribution of mesothe-lioma. Subsequently, however, Baris et al. (1987) sur-veyed Sarihidir and reported three cases of mesothelio-ma. Baris et al. also reported fiber characteristics from

the affected villages, indicating that zeolite fibers werepresent ubiquitously but other types of fibers varied be-tween villages.

The most disturbing implication of the observations inTurkey is that iferionite is indeed the cause ofthe highrates of mesothelioma, then erionite is capable of induc-ing mesothelioma in humans at low exposures. Baris etal. (1987) reported total fiber levels in the villages from0.004 to 0.175 fibers/ml, and these measurements in-cluded other dusts in addition to zeolite. Simonato et al.(1989) reported newer estimates offiber characteristics inKarain and Sarihidir (two of the affected villages) andfound levels to be 0.002-0.010 (-800/o zeolite) and 0.001-0.029 (- 600/o zeolite), respectively.

In vivo experiments have further demonstrated the highfibrogenic and carcinogenic potential of erionite-bearingdusts. Suzuki and Kohyama (Suzuki, 1982; Suzuki andKohyama, 1984, 1988) studied the effects on mice of in-trapleural injections of two erionite samples, one fromNeedle Peak, Nevada (Minerals Research; listed by Su-zuki and Kohyama as "Needle Park"), and one from anunknown locality (Resource International Company). Inmice injected intrapleurally with 2 mg of dust, NeedlePeak erionite induces tumors at a rate of 54.5ol0, com-pared with 0-25o/o for chrysotile and 40.50/o for "amosite"asbestos; fibrosis also develops after injection of any ofthe dusts. They also performed experiments using higherdoses, but a low percentage ofmice survived long enoughto develop tumors (>7 months). Maltoni et al. (1982) areinvestigating the effect of method of exposure to erioniteon the induction of mesotheliomas in rats. For their ini-tial results, they reported that erionite (sedimentary; ob-tained from G. Gottardi, University of Modena, Italy)induces mesotheliomas by intrapleural injection aI a raleof90o/o (nine often rats), whereas riebeckite asbestos in-duces mesothelioma by intraperitoneal injection at a rateof l00o/o (12 of l2).

Wagner and coworkers have also studied the in vivoeffects ofexposure to erionite using specific pathogen-freeSprague-Dawley rats (Wagner, 1982) and Fischer 344 rats(Wagner et al., 1985). They used intrapleural injection(20 mg/rat) and inhalation to study the carcinogenic po-tential of four erionite samples: Oregonian erionite (fromF. Mumpton, Minerals Research), nonfibrous syntheticzeolite (chemically identical to erionite, from R. Taylor,Laporte Industries), a New Znaland erionite (similar sizedistribution to the Oregonian sample, though fibers areslightly thicker), and a Turkish (Karain) rock determinedto consist of "poorly consolidated rock . . . [made ofl in-completely formed erionite . . . in an amorphous matrixwhich has the same composition as erionite" (Wagner etal., 1985). In the rats intrapleurally injected with Orego-nian erionite and Turkish rock, 40 of 40 and 38 of 40developed mesotheliomas, respectively, with mean sur-vival times of 390 and 435 d; the New Znaland erionitewas -Y+ as potent as the Oregonian and Turkish erio-nites. For comparison, in the same experiment, chrysotileinduced 19 mesotheliomas in 40 rats with a mean sur-


vival time of 678 d. The same effect was observed in theinhalation experiments: 27 of 28 rats exposed to Orego-nian erionite developed mesotheliomas with a mean sur-vival time of 580 d compared with one of 28 rats exposedto riebeckite asbestos (UICC standard) with a mean sur-vival time of 917 d. The synthetic zeolite was also testedin the inhalation experiments and induced two tumors in28 rats with a mean survival time of 784 d. With respectto the erionite sample, they stated "No other dusts wehave investigated have produced this high incidence oftumours particularly following inhalation" (Wagner et al.,1985). Coffin et al. (1989a) confirmed the observarionthat erionite-treated rats develop mesotheliomas at ahigher rate and in a shorter time than chrysotile asbestos-or riebeckite asbestos-treated rats; the erionite used wasfrom Rome, Oregon (Minerals Research), and was pre-pared by either HrO sedimentation or air elutriation.

In two later studies, the Wagner group confirmed theiroriginal finding. Johnson and Wagner (1989) exposed Fi-scher 344 rats to erionite from Rome, Oregon (obtainedfrom Minerals Research), by inhalation in a dust-rich en-vironment (10 mg/m3) and found that erionite exposureproduces both fibrosis and mesothelioma; three of thethree rats exposed to dust for 12 weeks and allowed torecover for l2 months developed mesothelioma. Hill etal. (1990) used intrapleural injection of Oregonian erio-nite in Porton rats to determine the dose-response rela-tionship for induction of mesotheliomas. They found asharp rise in mesothelioma rate from 0o/o mesotheliomasat 0.01 mg/rat to -90o/o at 1.0 mg/raI: they stated "erio-nite is over 200 times more tumourogenic than crocido-lite."

In vitro studies have demonstrated that erionite-bear-ing dusts are both cytotoxic and genotoxic. Poole et al.(1983b) studied the genotoxic effects ofOregonian erio-nite (Minerals Research) by monitoring morphologicaltransformations and unscheduled DNA repair in mouse-embryo fibroblasts (cells that reside in the connective tis-sue and that are responsible for collagen production, i.e.,that are involved in fibrosis). Erionite was found to beactive in both of these tests, whereas amphibole asbestosdoes not cause morphological transformations (Poole etal., 1983a). Numerous studies have also demonstratedthat erionite is cytotoxic (Palekar et al., 1988; Brown etal., 1989) and genotoxic (Palekar et al., 1987; 1989a,1989b) to Chinese-hamster lung cells. Brown et al. (1989)found that the cytotoxic effects are related to long, thinfibers, since milling of the sample to reduce the fiberlengths also reduces its activity. The cytotoxic and geno-toxic activities of erionite are slightly less than those ofasbestos when compared on a mass basis, but comparisonon a per-fiber basis shows that the activities of erioniteare much greater than those of asbestos (Palekar et al.,1988; Brown et al., 1989).

In other tests for genotoxicity, Hansen, Mossman, andcoworkers (Hansen and Mossman, 1987; Mossman et al.,1989; Mossman and Sesko, 1990) have shown that er-ionite (Rome, Oregon; from R. Davies) is as effective as

riebeckite asbestos in catalyzing the production of thesuperoxide anion from hamster and rat alveolar macro-phages. In contrast, however, Pezerat et al. (1989) foundthat though Oregonian erionite is nearly as effective asmost types of asbestos aI catalyzing the production of Oradicals from an aqueous medium, it is inactive whencompared with chrysotile (UICC standard B) or fibrousbrucite. Kelsey et al. (1986) found that erionite (Rome,Oregon; obtained from V. Timbrell) induces SCEs slight-ly in Chinese-hamster ovary cells, whereas riebeckite as-bestos (UICC standard) does not; ultraviolet light, how-ever, is much more effective at inducing SCEs. BothOregonian erionite and riebeckite asbestos (UICC stan-dard) induce low levels of chromosomal aberrations inthe Chinese-hamster ovary cells (Kelsey et al., I 986). Cof-fin et al. are attempting to relate the biological activity oferionite to its mineralogical characteristics (Cofrn et al.,1 989b).

Mordenite and other zeolites. In vivo experiments bySuzuki and Kohyama (Suzuki, I 982; Suzuki and Kohya-ma, 1984, 1988) suggest that a mordenite-bearing dustand zeolite 4Aare fibrogenic but noncarcinogenic. Suzukiand Kohyama used intraperitoneal injection in mice totest the biological response to various zeolites. Includedin their experiments was a mordenite-bearing sample(Resource International Company, Denver) that con-tained both granular and fibrous morphologies and a syn-thetic zeolite, 44. (Union Carbide Corporation); quanti-tative X-ray diffraction of the mordenite-bearing samplehas shown that it contains -63.50/o impurities, includingclinoptilolite, feldspar, opal-CT, and gypsum (Guthrie andBish, unpublished data). In fact, mordenite was used inthese experiments to test the relationship between parti-cle shape and activity among zeolites. The mordenitesample was described as a mixture of fibrous and nonfi-brous forms, despite the fact that mordenite is uniquelyfibrous. Hence, it should be recognized that their resultsapply to an impure mordenite sample. Their experimentsshow that lO-mg doses of either the mordenite-bearingsample or zeolite 4A induce no tumors in mice for ex-periments up to 23 months in duration; fibrosis, however,does result from the exposure. On the other hand, thesame experiments showed that 0.5- and 2.0-mg doses oferionite (Needle Peak, Nevada) induce tumors at 33.3and 54.5o/o, respectively; also, fibrosis in erionite-exposedmice is more pronounced. In their group exposed to a l0-mg dose of erionite, a 37 .5o/o rate of tumor induction wasobserved, but only eight rats survived to > 7 months, i.e.,exposure to erionite may elicit a dose-dependent responsein the lungs of mice, but this relationship cannot be testedbecause of the poor statistical significance of the resultsfrom the group exposed to high concentrations of erio-nite.

Maltoni and Minardi (1988, 1989) studied the biolog-ical activity of synthetic zeolites used in detergents (MS44' and MS 5A, Na and Ca rich, respectively; source notgiven) by intraperitoneal, intrapleural, and subcutaneousiniection in rats. For both zeolites and all routes of ex-


posure, tumors appeared but not at rates significantly dif-ferent from those observed in the control groups that wereinjected with HrO (-25-50o/o). In the same assay, how-ever, riebeckite asbestos induces tumors following injec-tion into the abdominal cavity at a tate of 97.5o/o.

In vitro experiments suggest that mordenite-bearingdust is much less active than erionite. Hansen, Mossman,and coworkers (Hansen and Mossman, 1987; Mossmanet al., 1989; Mossman and Sesko, 1990) found that amordenite-bearing sample (source not given) is less effec-tive than sepiolite and much less effective than erioniteat stimulating the release of the superoxide anion fromrat alveolar macrophages. Palekar et al. (1988) found thatthis same mordenite is noncytotoxic to Chinese-hamsterlung cells. Quantitative X-ray diffraction of this morden-ite-bearing sample has shown that it contains -50.50/oimpurities, including clinoptilolite, feldspar, and opal-CT(Guthrie and Bish, unpublished data). As was the case forthe in vivo experiments of Suzuki and Kohyama, mor-denite was used as a nonfibrous-zeolite control in the invitro assays, despite that mordenite is uniquely fibrous.

Rom et al. (1983) discussed the implications of fibrouszeolite health hazards with respect to the western UnitedStates and stressed the need for epidemiological studiesin this region.

DrscussroN

The wide range of minerals that have been studied byvarious techniques offers the potential for revealing thecauses of a mineral's biological activity. Indeed, it is clearfrom the dusts studied already that minerals exhibit dif-ferent activities and elicit different biological responses(Table 2). In fact, differences in biological response canbe found both between mineral species and between dif-ferent samples of the same mineral species. The varia-tions in response likely reflect variations in the interac-tions between the mineral surfaces and biologicalcomponents (i.e., cell surfaces, enzymes, proteins, DNA,etc.). Ideally, if this observed variation can be related todifferences in the physical and chemical properties oftheminerals, then the mechanisms of mineral toxicity maybe elucidated. Unfortunately, several mineralogical prob-lems are present in the studies reviewed above that makesuch inferences difficult if not impossible. Generally, theprimary mineralogical aspects that are controlled in mostbiological experiments are the particle shape and size dis-tribution and mass concentration or dose employed, sincethese parameters appear to relate to the material's bio-logical activity as determined by in vivo methods (e.g.,Stanton et al., l98l). The exact mineral content of thedusts, however, is rarely characterized. In other words,little attention is generally given to the identification andamount of contaminants in the dust sample. Instead, itis assumed that the mineral content of the sample match-es the information provided by the supplier. However,samples obtained from most suppliers potentially containa mixture of minerals and often are simply a differentmineral from the one listed on the label. The mordenite

TreLe 2. Summary of data on the biological activities of claysand zeolites

EpidemioMineral logical ln vivo In vitro

HematiteGoethite

LepidocrociteBoehmiteFibrous bruciteKaoliniteHalloysiteAntigoriteBerthierineChlorite (Fe-rich)TalcMica/micajike

craysSepiolite

Palygorskite

Erionite

Mordenite

Zeolites 4A and 5A

n.o.

n.d.n.d.n.d.+n.o.n.o.n-o.n.d.- t o ++

- t o +

+ + +

(0

(0

+ t o + + ( 0+ (t)+ to ++ (f)- t o + + ( 0+++ ( t )- t o + + ( 0- t o + ( t )n.d.n.d.n.d.

- t o + ( D

n.d.

n.d.n.d.T T

+ to +++n.o.

+ + ++ + +- to +++- t o +

+ + + ( D - t o + + +- (t)- to +++ ( f ) - to +++- to +++ (t)+ + + ( 0 + + ++++ ( t )+ ( f ) - t o +- (r)+ (0 n.d.- (t)

Note; Symbols: - indicates inactive; + indicates active; n'd. indicatesno data availade; f and t indicate fibrogenic and tumorigenic, respectively.

samples used in both the in vitro experiments (Hansenand Mossman. 1987: Palekar et al.. 1988: Mossman etal., 1989; Mossman and Sesko, 1990) and the in vivoexperiments (Suzuki, 1982; Suzuki and Kohyama, 1984,1988) illustrate this well. Each sample actually containsa mixture of mordenite, clinoptilolite, feldspar, opal-CT,and gypsum (Guthrie and Bish, unpublished data). Thepublished mordenite-toxicity data actually apply to amixture of minerals that is s50o/o mordenite.

Another mineralogical problem in biological studies isthat the surface properties of the samples are generallynot adequately characterized. Recent work (e.g., Pezeral,1990) has suggested that although the fibrous shape ofamaterial may be important in maintaining the particle inthe target organ or in enhancing surface area, the mech-anisms by which minerals are toxic relate to their surfaceproperties, such as active oxidation/reduction sites. In-deed, the activity of a mineral varies with surface state(Nolan et al., l99l) and surface area (Gormley and Ad-dison, 1983), which in turn can vary substantially be-tween samples. Thus, it is not only important to controlthe surface aspects of a mineral during an experiment,but it is important to characteize these properties so thatthey can be related to biological activity.

These mineralogical deficiencies in biomedical re-search can be rectified through collaborative efforts be-tween minerals scientists and health scientists. Such col-laboration should involve both characterization of themineralogical aspects of the experiment and design of ex-periments that will allow mechanistic questions to be ad-dressed. For example, an amphibole has numerous prop-

n.d.

n.o.


erties that might contribute to its activity (e.g., brokenSi-O bonds, "exchangeable" cations in the A site, poly-valent cations in the octahedral sites, underbonded O re-sulting from Al substitution in the tetrahedral sites, spe-cific surface periodicities). Hence, the results of a studyon amphibole-induced pathogenesis may record effectsfrom several mineralogical properties. On the other hand,it is possible to isolate the effects of a specific mineral-ogical characteristic by an appropriate choice ofmineralpairs. For instance, the role ofpolyvalent cations in theoctahedral site can be determined by comparing the ac-tivities between two minerals that differ only in the com-position of the octahedral site (e.g., tremolite and ferro-actinolite; annite and phlogopite). This type ofapproachcould be extremely effective for determining mineralogi-cal mechanisms of mineral-induced pathogenesis. Suchinformation will lead to both a better understanding ofdiseases such as cancer and more effective regulation ofminerals, since regulations can be based on mineralogicalproperties additional to particle size and shape.

Furthermore, it should be recognized that though amineral is active in a particular assay, it may pose limitedrisk to humans. A complete understanding of the numer-ous factors that contribute to mineral-induced pathogen-esis is essential before the results ofany one assay can beused to predict risk to humans.

AcrNowr,nncMENTS

I would like to thank D.L. Bish, G.D. Guthrie, Sr., and C.S. Nicholson-Guthrie for extensive discussions and encouragement during the prepa-ration of this manuscript. I also benefrted from discussions with F.Mumpton and B. l€hnert. Thoughtful reviews of the manuscript wereprovided by D.L. Bish, J. Hughes, B. I-ehnert, W. Moll, B.T. Mossman,M. Ross, and D. Yaniman My time was supported by a postdoctoralfellowship from the Director's office at the Los Alamos National Labo-ratory.

RnrnnnNcrs crrEDAchard, S., Perderiset, M., and Jaurand, M.-C. (1987) Sister chromatid

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