effects of formaldehyde, acetaldehyde, benzoyl …...effects of formaldehyde, acetaldehyde, benzoyl...

6
[CANCER RESEARCH 45, 2522-2526, June 1985] Effects of Formaldehyde, Acetaldehyde, Benzoyl Peroxide, and Hydrogen Peroxide on Cultured Normal Human Bronchial Epithelial Cells Andrew J. Salariino,1 James C. Willey,2 John F. Lechner,3 Roland C. Grafstrom,4 MoìraLaVeck, and Curtis C. Harris5 Laboratory of Human Carcinogenesis, D/ws;on of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20205 ABSTRACT The effects of several aldehydes and peroxides on growth and differentiation of normal human bronchial epithelial cells were studied. Cells were exposed to formaldehyde, acetaldehyde, benzoyl peroxide (BPO), or hydrogen peroxide (HPO). The effect of each agent on the following parameters was measured: (a) clonal growth rate; (b) squamous differentiation; (c) DNA dam age; (d) ornithine decarboxylase activity; (e) nucleic acid synthe sis; (f) aryl hydrocarbon hydroxylase activity; and (g) arachidonic acid and choline release. None of the agents were mitogenic, and their effects were assessed at concentrations which reduced growth rate (population doublings per day) to 50% of control. The 50% of control concentrations for the 6-h exposure were found to be 0.065 ITIM BPO, 0.21 HIM formaldehyde, 1.2 mw HPO, and 30 mw acetaldehyde. BPO-exposed cells were smaller than controls (median cell planar area, 620 sq ^m versus 1150 sq tim), and acetaldehyde-exposed cells were larger than con trols (median cell planar area, 3200 sq um). All agents increased the formation of cross-linked envelopes and depressed RNA synthesis more than DNA synthesis. HPO caused DNA single- strand breaks, while formaldehyde and BPO caused detectable amounts of both single-strand breaks and DNA-protein cross links. Other effects included increased arachidonic acid and choline release due to HPO. The similarities and differences of the effects of these aldehydes and peroxides to those caused by tumor promoters are discussed. INTRODUCTION Recent advances in techniques for obtaining replicative cul tures of NHBE6 cells grown in serum-free medium (25, 29) have made it possible to investigate the response of these cells to a variety of naturally occurring and synthetic substances that may participate in the causation of human lung cancer. Aldehydes and peroxides are of particular interest because of their presence 1 Present address: Baltimore Veterans Administration Medical Center, Baltimore, MD21218. 1 Present address: Pulmonary Branch, Heart, Lung, and Blood Institute, Be thesda. MO 20205. * Present address: Culture Media Branch, Centers for Disease Control, Atlanta, GA 30333. 'Present address: Department of Toxicology, Karolinska Institutet, S-104 01 Stockholm .60, Sweden. *To whom requests for reprints should be addressed, at Building 37, Room 2C05, National Cancer Institute, NIH, MD 20205. • The-abbreviations used are: NHBE, normal human bronchial epithelial; FMD, formaldehyde; ACT, acetaldehyde; BPO, benzoyl peroxide; HPO, hydrogen per oxide; ODC, ornithine decarboxylase: ARA, arachidonic acid; CH, choline; CLE, cross-linked envelope; SSB, single-strand breaks; DPC, DNA-protein cross-links; SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ID«,, concentration which reduced growth rate (population doublings per day) to 50% of control. Received 6/13/84; revised 1/9/85; accepted 1/31/85. in the environment, e.g., tobacco smoke, and their potential role in tumor promotion (11). The present report describes the acute pathobiological effects of FMD, ACT, BPO, and HPO utilizing this in vitro model. FMD is ubiquitous (21, 33). It has been shown to be a weak promoter in C3H/1 OTVa cells (15) and to cause nasal passage cancers in rats (41). ACT is present in cigarette smoke in higher concentrations than is FMD [800 versus 30 ^g/cigarette, respec tively (9, 45)], enhances the tumorigenic potency of benzo(a)- pyrene in the hamster lung (12), and also causes nasal passage cancers in rats.7 HPO is endogenously formed in cells as a consequence of free radical formation and can be increased by exogenous agents such as ionizing radiation (24) and TPA (17). HPO affects the primary and secondary structure of DNA in vitro (31, 36) and increases the frequency of sister chromatid exchanges in Chinese hamster V79 cells (6). However, its mutagenic activity in mammalian cells is low (7). HPO does not appear to be tumorigenic in rats8 but may enhance methylazoxymethanol acetate-induced small intestinal adenocarcinoma (20). In initia tion-promotion experiments using the mouse skin carcinogenesis model, BPO and HPO each have tumor-promoting activity (22, 39). A higher-than-expected incidence of lung cancer has been reported in factory workers exposed to chemical dust containing BPO and its precursors (38). Parameters chosen for study in the present investigation were selected because of their relationship to cellular growth and control of differentiation and because of alterations reported previously in other investigations (5, 7, 13, 14, 16, 34, 35, 40, 46). MATERIALS AND METHODS Cells and Media. Adult tracheobronchial tissues were obtained from human donor autopsy specimens and maintained in culture according to methods described previously (25, 26). Growth and morphological ex periments utilized cells from 8 donors, biochemical experiments, 12 donors, and CLE studies, 4 donors. Primary epithelial cell cultures were enzymatically dissociated with calcium-free 4-(2-hydroxyethyl)-1 -pipera- zineethanesulfonic acid-buffered 0.9% NaCI solution (saline) containing EDTA, 0.02%, polyvinylpyrrolidine, 1%, and trypsin, 0.02% (25), and subcultured into surface-treated Costar Pétri dishes [fibronectin, 10 ¿»g/ ml, bovine serum albumin, 10 ^g/ml, and Vitrogen collagen, 30 ^g/ml (26)]. Two types of serum-free media were used for these experiments, a "growth" medium [LHC-1 medium (25)] supplemented with triiodothy- ronine and pituitary extract (29) and a maintenance medium that lacked these supplements and epidermal growth factor. Maintenance medium was used to determine whether any of the test agents could substitute for absent growth factors and to provide an environment wherein low 7V. J. Feron, personal communication. " S. Takayama, personal communication. CANCER RESEARCH VOL. 45 JUNE 1985 2522 on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Upload: others

Post on 18-Apr-2020

21 views

Category:

Documents


0 download

TRANSCRIPT

[CANCER RESEARCH 45, 2522-2526, June 1985]

Effects of Formaldehyde, Acetaldehyde, Benzoyl Peroxide, and Hydrogen

Peroxide on Cultured Normal Human Bronchial Epithelial Cells

Andrew J. Salariino,1 James C. Willey,2 John F. Lechner,3 Roland C. Grafstrom,4 MoìraLaVeck, andCurtis C. Harris5

Laboratory of Human Carcinogenesis, D/ws;on of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20205

ABSTRACT

The effects of several aldehydes and peroxides on growth anddifferentiation of normal human bronchial epithelial cells werestudied. Cells were exposed to formaldehyde, acetaldehyde,benzoyl peroxide (BPO), or hydrogen peroxide (HPO). The effectof each agent on the following parameters was measured: (a)clonal growth rate; (b) squamous differentiation; (c) DNA damage; (d) ornithine decarboxylase activity; (e) nucleic acid synthesis; (f) aryl hydrocarbon hydroxylase activity; and (g) arachidonicacid and choline release. None of the agents were mitogenic,and their effects were assessed at concentrations which reducedgrowth rate (population doublings per day) to 50% of control.The 50% of control concentrations for the 6-h exposure were

found to be 0.065 ITIM BPO, 0.21 HIM formaldehyde, 1.2 mwHPO, and 30 mw acetaldehyde. BPO-exposed cells were smaller

than controls (median cell planar area, 620 sq ^m versus 1150sq tim), and acetaldehyde-exposed cells were larger than con

trols (median cell planar area, 3200 sq um). All agents increasedthe formation of cross-linked envelopes and depressed RNAsynthesis more than DNA synthesis. HPO caused DNA single-

strand breaks, while formaldehyde and BPO caused detectableamounts of both single-strand breaks and DNA-protein cross

links. Other effects included increased arachidonic acid andcholine release due to HPO. The similarities and differences ofthe effects of these aldehydes and peroxides to those causedby tumor promoters are discussed.

INTRODUCTION

Recent advances in techniques for obtaining replicative cultures of NHBE6 cells grown in serum-free medium (25, 29) have

made it possible to investigate the response of these cells to avariety of naturally occurring and synthetic substances that mayparticipate in the causation of human lung cancer. Aldehydesand peroxides are of particular interest because of their presence

1Present address: Baltimore Veterans Administration Medical Center, Baltimore,

MD21218.1Present address: Pulmonary Branch, Heart, Lung, and Blood Institute, Be

thesda. MO 20205.* Present address: Culture Media Branch, Centers for Disease Control, Atlanta,

GA 30333.'Present address: Department of Toxicology, Karolinska Institutet, S-104 01

Stockholm .60, Sweden.*To whom requests for reprints should be addressed, at Building 37, Room

2C05, National Cancer Institute, NIH, MD 20205.•The-abbreviations used are: NHBE, normal human bronchial epithelial; FMD,

formaldehyde; ACT, acetaldehyde; BPO, benzoyl peroxide; HPO, hydrogen peroxide; ODC, ornithine decarboxylase: ARA, arachidonic acid; CH, choline; CLE,cross-linked envelope; SSB, single-strand breaks; DPC, DNA-protein cross-links;SDS, sodium dodecyl sulfate; TPA, 12-O-tetradecanoylphorbol-13-acetate; ID«,,concentration which reduced growth rate (population doublings per day) to 50% ofcontrol.

Received 6/13/84; revised 1/9/85; accepted 1/31/85.

in the environment, e.g., tobacco smoke, and their potential rolein tumor promotion (11). The present report describes the acutepathobiological effects of FMD, ACT, BPO, and HPO utilizingthis in vitro model.

FMD is ubiquitous (21, 33). It has been shown to be a weakpromoter in C3H/1 OTVacells (15) and to cause nasal passagecancers in rats (41). ACT is present in cigarette smoke in higherconcentrations than is FMD [800 versus 30 ^g/cigarette, respectively (9, 45)], enhances the tumorigenic potency of benzo(a)-

pyrene in the hamster lung (12), and also causes nasal passagecancers in rats.7

HPO is endogenously formed in cells as a consequence offree radical formation and can be increased by exogenous agentssuch as ionizing radiation (24) and TPA (17). HPO affects theprimary and secondary structure of DNA in vitro (31, 36) andincreases the frequency of sister chromatid exchanges inChinese hamster V79 cells (6). However, its mutagenic activityin mammalian cells is low (7). HPO does not appear to betumorigenic in rats8 but may enhance methylazoxymethanol

acetate-induced small intestinal adenocarcinoma (20). In initiation-promotion experiments using the mouse skin carcinogenesismodel, BPO and HPO each have tumor-promoting activity (22,39). A higher-than-expected incidence of lung cancer has been

reported in factory workers exposed to chemical dust containingBPO and its precursors (38).

Parameters chosen for study in the present investigation wereselected because of their relationship to cellular growth andcontrol of differentiation and because of alterations reportedpreviously in other investigations (5, 7, 13, 14, 16, 34, 35, 40,46).

MATERIALS AND METHODS

Cells and Media. Adult tracheobronchial tissues were obtained fromhuman donor autopsy specimens and maintained in culture according tomethods described previously (25, 26). Growth and morphological experiments utilized cells from 8 donors, biochemical experiments, 12donors, and CLE studies, 4 donors. Primary epithelial cell cultures wereenzymatically dissociated with calcium-free 4-(2-hydroxyethyl)-1 -pipera-zineethanesulfonic acid-buffered 0.9% NaCI solution (saline) containing

EDTA, 0.02%, polyvinylpyrrolidine, 1%, and trypsin, 0.02% (25), andsubcultured into surface-treated Costar Pétridishes [fibronectin, 10 ¿»g/

ml, bovine serum albumin, 10 ^g/ml, and Vitrogen collagen, 30 ^g/ml(26)]. Two types of serum-free media were used for these experiments,a "growth" medium [LHC-1 medium (25)] supplemented with triiodothy-

ronine and pituitary extract (29) and a maintenance medium that lackedthese supplements and epidermal growth factor. Maintenance mediumwas used to determine whether any of the test agents could substitutefor absent growth factors and to provide an environment wherein low

7V. J. Feron, personal communication." S. Takayama, personal communication.

CANCER RESEARCH VOL. 45 JUNE 1985

2522

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTSOF ALDEHYDESAND PEROXIDESON HUMAN CELLS

rates of cell division were occurring.Clonal Growth Assay. Five thousand cells were inoculated into 60-

mm dishes, allowed 24 to 48 h to equilibrate with their environment, thenexposed for either 6 h or continuously to test agents. After 7 to 9 daysof incubation, the cultures were fixed, stained, and number of cells percolony counted using an ARTEK 800 image analyzer (25,43). To obtainthe mean clonal growth rate (population doublings per day), the numberof cells in 9 randomly selected clones from each of 2 dishes was countedand the log: of the average number of cells per clone was divided by thenumber of days of incubation (10, 25, 28). Variance-weighted Student's

t test was used to evaluate significant differences between control andtreated groups.

Sources of Test Agents. Chemicals were obtained from the followingsources: acetaldehyde, benzoyl peroxide, and formate, Aldrich ChemicalCompany, Milwaukee, Wl; formaldehyde. Mallinckrodt, St. Louis, MOand Fisher Scientific Company, Fair Lawn, NJ; and hydrogen peroxide,

Fisher.Biochemical Assays. For each assay, cells were plated at a density

of 50,000/sq cm in a 24-well cluster dish. Unless otherwise specified, all

incubations with test agents were for 6 h.ARA and CH Release. Cell cultures were incubated in medium

containing 1 ¿iCi/mlof either [5,6,8,9,11,12,14,15-3H]ARA (specific activity, 86.6 Ci/mmol; New England Nuclear, Boston, MA) or [mefny/-3H]CH

(80 Ci/mmol; New England Nuclear) 24 h prior to exposure to the testagents. Subsequently, cells were rinsed 3 times to remove unincorporated radioactive substrate, and 0.25 ml of new medium containing thetest agent was added. After incubation with test agents, media wereremoved, and radiolabeled ARA and CH liberated from membranes intothe medium were measured (13).

Aryl Hydrocarbon Hydroxylase Activity. After incubation with testagents, cells were incubated for an additional 6 h with [3H]benzo(a)-

pyrene, 32 j/Ci/ml, (20 Ci/mmol; Amersham/Searie Corporation, ArlingtonHeights, IL), and the 3H2O (formed by the NIH shift reaction) was

separated from benzo(a)pyrene by an activated charcoal column andmeasured (44).

ODC Activity. After incubation with test agents, a cell lysate wasprepared by subjecting cultures to 3 sequential freeze-thaw cycles. Thecell lysate was incubated for 1 h with i_-[1-14C]omithine hydrochloride,

100 nCi/ml (58 mCi/mmol; Amersham-Searle), and the liberated metabolic "C02 was trapped in NaOH-soaked paper discs and quantified by

scintillation counting (30).CLE Assay. A modification of the method of Sun and Green (40) was

used to determine the percentage of cells capable of forming CLEs. Cellswere subcultured at a density of 100,000 cells/10 sq cm. These cellswere then incubated in 1.5 ml of the agarose medium mixture (44) towhich test agents had been added. At the end of the 6-h incubation

period, the average number of cells per x 100 field was determined from10 to 20 such fields with a phase contrast microscope. Then 1.5 ml of asolution of 4% SOS and 20 HIM dithiothreitol dissolved in 4-(2-hydroxy-ethyl)-l-piperazineethanesulfonic acid-buffered saline was added overthe agarose medium gel. After 3 to 6 h of further incubation at 37°C,the

average number of CLEs per x 100 field, determined as described

above, was divided by the average number of cells per field.DNA and RNA Synthesis. Cells were incubated for 48 h in a medium

supplemented with [14C]thymidine, 10 nCi/ml (specific activity, 50 mCi/

mmol; New England Nuclear), rinsed with nonradioactive medium, andincubated in medium containing test agents. Subsequently, culturesreceived a 2-h pulse of either [3H]thymidine, 6 /¿Ci/ml(specific activity,72 Ci/mmol; New England Nuclear), or [3H]uridine, 8 /iCi/ml (specific

activity, 26 Ci/mmol; New England Nuclear) for assessment of DNA andRNA synthesis, respectively. Cultures were then rinsed with phosphate-

buffered saline, dissolved in 0.2 N NaOH containing calf thymus DNA, 40Mg/ml, or 2% SDS containing bovine serum albumin, 10 ^g/ml (for RNA),poured onto acid-soaked glass fiber filters, and washed with 95%

ethanol. Radioactivity was then measured (25).DNA Damage Assays. The alkaline elution technique was used to

quantify SSB or DPC (23). Cells were incubated with test agents for 1h, then removed with a rubber policeman at 4°C in a solution ofDulbecco's Ca- ':Mg?*-free phosphate-buffered saline containing 15 mw

EDTA and collected onto a 2-itm polycarbonate filter (Nuclepore Corpo

ration, Pleasanton, CA) to measure DNA SSB. The cells were lysed with5 ml of 2% SDS:0.1 M glycine:0.02 M EDTA (pH 9.6), and 2 ml of thesame solution containing proteinase K, 0.5 mg/ml, was then pumpedthrough the filter at 0.04 ml/min. This solution was followed by 0.02 MEDTA (acid form):0.1% SDS, plus tetrapropylammonium hydroxideadded to give a final pH of 12.2 (14,18). Eluted fractions were collectedand assayed for radioactivity. The combination of the polycarbonatefiltration proteinase K digestion and the addition of SDS in the elutingsolution completely removed the DPC effect of the different agents atthe indicated concentrations tested. An internal standard of [3H]thymi-

dine-labeled L1210 cells that had received 300 rads of ionizing radiationat 0°Cwas included in each assay. To measure the extent of DPC, the

assay was modified by omission of the proteinase digestion of the celllysates, and SDS was deleted from the eluting solution. The cells wereexposed to 3 krads of ionizing radiation (to shear cellular DNA) beforecollecting these cells on polyvinyl chloride filters (Millipore Corporation,Bedford, MA). The SSB or DPC frequencies were estimated and calculated as described by Kohn et al. (23).

Morphological Quantitäten. Cultures were fixed with a 1% FMD-4%

glutaraldehyde solution (32) for light and electron microscopy. For lightmicroscopic studies, cultures were stained with May-Grunwald Giemsa

stain. Cell planimetry was performed with the aid of the image analyzer(26,27).

RESULTS

Cell Proliferation. The effects of various concentrations ofFMD, ACT, BPO, and HPO on clonal growth rate of the NHBEcells are shown in Table 1. All compounds inhibited growth, andthe inhibition was dose dependent. Except for HPO, these agentswere more inhibitory when cells were incubated in growth medium. The order of inhibitory potency of the agents was thesame for either 6- or 24-h exposure. Formate, a nontoxic inter

mediary metabolite of FMD, had little effect on cell replication upto 30 rnw. Cultures were most sensitive to BPO and FMD [IDso,

Table 1

Effects of aldehydes and peroxides on clonal growth rates of NHBE cells

Cells were exposed to the test agent for 6 h and subsequently allowed to growin the indicated medium for 7 to 9 days. Growth medium consisted of LHC-1 (25)supplemented with tniodothyronme and pituitary extract. Maintenance mediumlacked these supplements as well as epidermal growth factor. Clonal growth rates(population doublings per day) were 0.45 ±0.05 for maintenance medium and 0.9±0.1 for growth medium. Duplicate dishes were counted on an image analyzer.Experiments were repeated 4 times.

AgentsACTFMDFormateBPOHPOConcentration(m«)0.33.030.00.0030.0300.3000.33.030.00.0030.0300.3000.0250.2502.500Culture

medium(%)Maintenance100

±3a94

±373±7100

±186±854±696±190±290

±2100±386±951

±1087±1500Growth99

±290±554±896±287

±940±12100

±199±294±990±360±1513±1096

±184±925

±131Mean ±SD.

CANCER RESEARCH VOL. 45 JUNE 1985

2523

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF ALDEHYDES AND PEROXIDES ON HUMAN CELLS

Table 2Effects of certain aldehydes and peroxides on release of ARA and CH and on activities of ODC and AHH* in

NHBE cells cultured in maintenance or growth mediumAHH values of control cells grown in M and G media were 22 ±3 and 14 ±4 pmol of [3H]benzo(a)pyrene

hydrolyzed per h per mg protein, respectively; ODC values of cells grown in M and G media were 0.13 ±0.2and 1.2 ±0.5 nmol i -|1-14Clornithinehydrolyzed per mg protein, respectively. Duplicatewells were measured

for each experiment. Three experimentswere done per compound. Basalvalues for ARA liberatedwith 15,800±2,330 cpm in M, 10,780 ±2,200 cpm in G, and for choline 3,878 ±610 cpm in M and 5,988 ±1,212 cpmm G

AgentsACTFMDFormateBPOHPOConcentration(rtiM)300.21300.0561.2%

of controlvalueMediumMGMGMGMGMGARA77±11"'C82

±2577±2367

±7°115

±2299±24106±19110

+10240±35e567±108e'0CH70±7C74±6C93

±997±2109±6104±296±296

±2174±3c'd146

±8eAHH63

±18e75±10e63±18e67±10e135

±18121±31100±18109±10161

±70115±33ODC47

±23e61±38°245

±60e191±25e24

±5°22±5C153

±62119±507

±5°36±12e

* AHH, aryl hydrocarbon hydroxylase; M, maintenance;G, growth.6 Mean ±SD.c Significantlydifferent from control, at P < 0.05, Student's t test.

Significantlydifferent from maintenanceand/or growth medium.

13.6 Mg/ml (56 UM) and 6.3 ^g/ml (210 MM), respectively], lesssensitive to HPO |ID6n, 40.8 i<g/ml (1.2 mu)], and at leastsensitive to ACT [IDso, 1.32 mg/ml (30 HIM)].

Biochemical Changes. The effects of these agents on NHBEcells after 6 h of exposure at their respective ID50sare shown inTable 2. HPO increased the amount of ARA and CH releasedinto the culture medium. The increased ARA release was mostpronounced in growth medium, while the increased CH releasewas greatest in maintenance medium (P < 0.05). The ratios ofARA to CH released by cells in maintenance and growth mediawere 1.4:1 and 3.9:1, respectively.

ODC activity was maximal at 200 ftu FMD in growth mediumand between 200 and 400 fiM for maintenance medium (data notshown). Thus, the IDso concentration was similar to the dosefound to be optimal for enhanced ODC activity. Significant decreases in various enzyme activities were also noted after 6 h ofexposure (Table 2). HPO profoundly decreased ODC activity inboth media but was more potent in maintenance medium.

FMD and ACT each inhibited aryl hydrocarbon hydroxylaseactivity. The other agents did not have a significant effect. Theeffects of these compounds on DMA and RNA synthesis after 6h of incubation in growth medium are shown in Table 3. HPO,BPO, FMD, and ACT each inhibited RNA synthesis more thanDNA synthesis at the IDso concentrations tested. These compounds also depressed DNA synthesis; when IDsovalues werecompared, FMD, HPO, and ACT were more potent than BPO.

Morphological Changes. The results of image analysis ofNHBE cell cultures are shown in Table 4. The planar surfacearea of NHBE cells that are capable of division was found to bebetween 1000 and 1500 sq /»m.A larger percentage of thesecells was found in growth medium than in maintenance medium.9

After 7 to 9 days of exposure to a 6-h IDsopulse, median planar

cell area was approximately 50% of controls for BPO and 300%of controls for ACT. While cells incubated in FMD, formate, andHPO tended to be smaller, they were not significantly differentfrom controls.

Table 3

Effects of certain aldehydes and peroxides on nucleic acid synthesis in culturedNHBE cells

All values are statistically different from control value, at P < 0.05 by Student's

f test. Duplicate wells were measured for each experiment. Experiments wererepeated 3 times. Typical values for 3H: "C control ratios were DNA, 5,504: 284dpm = 19; RNA, 16,642:167 = 99.

% of control value

AgentConcentration Thymidine incorpora-

(rriM) tion Uridineincorporation

ACTFMDBPOHPO300.210.0561.234 ±7"24

±544±534±714±514±414±511

±3' Mean ±SD.

Table 4

Effects of certain aldehydes and peroxides on median size and CLE formation ofNHBE cells

Cells were cultured in growth medium and incubated for 6 h with IDsoconcentrations of the test agents. Duplicate dishes were measuredon an image analyzerto obtain cell sizes. Each experiment was repeated 3 times. CLE in duplicate wellswere counted visually. Experiments were also repeated 3 times.

AgentConcentration

(DIM) Median cell size (sq *>m) CLE(%)

NoneACTFMDFormateBPOHPOA23187300.21300.0561.210-"1150±400*3200±680"983

±400925±400620±70"1030

±350Notdone2

±0.57±2.0"12

±2.063+0.815

±3.0"18±4.0"23

±3.0*

•A. J. Saladmo,unpublished results.

4 Mean ±SD." Statistically significantby Student's t test, at P < 0.05, compared with control

values.

In the present studies, NHBE cells exhibited a low but constantrate of CLE formation of approximately 2.0% (Table 4); nosignificant difference was found between maintenance andgrowth media (24 h after subculture). CLE formation after addition of formate was found to be identical to that of controls. Allthe other compounds caused significantly greater CLE formationthan controls; HPO and BPO were more effective than was FMD.

CANCER RESEARCH VOL. 45 JUNE 1985

2524

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF ALDEHYDES AND PEROXIDES ON HUMAN CELLS

TablesDNA SSB and DPC caused by various agents in human bronchial epithelial cells

Cells were exposed to the indicated agents for 1 h in serum-free medium andsubsequently assayed for DNA damage by the alkaline elution technique. Bronchialcells were labeled with [14C]thymidine; L-1210 cells that had received 300 radswere labeled with [3H]thymidine and used as internal standard. The extent of SSBor DPC per 10'°dallons was expressed and calculated as described by Kohn et

al. (23).

AgentsACT

FMDForniateBPOHPOConcentration

(mM)1

0.110.10.1SSB<1.0

1.4<1.0

4.927.8DPC<2.0

10.8<2.0

5.3<2.0

ONA Damage. The potency of the tested compounds to causeDNA damage was measured by the sensitive alkaline elutiontechnique (Table 5). Exposure of cells to HPO, BPO, or FMD at100 MMconcentrations for 1 h caused significant levels of SSBor DPC. Whereas HPO caused primarily SSB, FMD causedsubstantially higher levels of DPC than SSB. Similar frequenciesof both types of DNA lesions were induced by BPO. In contrast,neither ACT nor formate up to 1 mw concentration induced anydetectable DNA damage.

DISCUSSION

The aldehydes and peroxides used in this study have beenshown to enhance the development of malignant cells in someanimal (12, 20, 39) and In vitro (15) models. It was therefore ofinterest to determine their acute effects on NHBE in vitro and tocompare these effects with those reported previously for TPA(43, 45, 46).

Formation of cross-linked envelopes occurs as a late event in

the terminal differentiation of epithelial cells. In the skin, thedistinctive CLE microscopic structure and precursors, e.g., in-

volucrin, are found only in the upper, nondividing levels of theepidermis (42). Furthermore, in cultured human epidermal cells,involucrin and its mRNA are found in the nonproliferative cells.In the present study, CLE formation was increased withoutsignificant increase in planar cell area. In contrast, both TPA andblood-derived serum have been observed to increase CLE for

mation, but they also each change NHBE cellular morphologyfrom small fusiform to large squamoid cells (26, 43). CLE formation may have occurred through one or more pathways. CLEformation is a calcium ion-dependent process (37) involving thecovalent cross-linking of the soluble protein monomer, involucrin

(39), by epithelial transglutaminase (1), and calcium ionophore isknown to induce CLE formation (42) in epidermal kerotinocytecultures and in NHBE (43). Although no information is availablefor bronchial epithelium, aldehydes and peroxides are known toeffect calcium homeostases in isolated hepatocytes (2). Inhibitionof membrane-bound Ca2+-ATPase (3) or release of Ca2+ from

intracellular compartments caused by peroxides has been shownto result in elevated cytoplasmic calcium levels (4). Furtherstudies of the effects of aldehydes and peroxides are needed todetermine if similar mechanisms regulate terminal differentiationof normal epithelial cells. Based on the above results, we speculate that the mechanism of CLE formation after aldehydes andperoxides is similar to calcium ionophore and different from TPA

and blood-derived serum.

Aldehydes and peroxides have been shown to cause DNAdamage, including SSB and DPC in mammalian cells (6, 7, 14,18). In NHBE cells, FMD, HPO and BPO each caused a distinguishing pattern of DNA damage (Table 5). FMD produced moreDPC than SSB, whereas HPO was more effective in causingSSB. The frequencies of SSB and DPC were similar in NHBEcells exposed to BPO. As reported previously in studies usingChinese hamster V-79 cells (6, 7), the quantitative relationships,

if any, between these DNA lesions and biological consequences,i.e., mutagenicity and cytotoxicity, are not apparent. For example, HPO produces a large number of SSB, and frans-platinum

produces a large number of DPC without either being detectablymutagenic. In addition to causing SSB and DPC in NHBE cells,FMD can inhibit DNA repair processes, i.e., the ligation steps inrepair of SSB caused by X-irradiation (18) and activity of O6-

alkylguanine DNA transalkylase (19). FMD has also been shownto be mutagenic in normal human cells and increases the mutagenicity of A/-methyl-A/-nitrosourea in a synergistic manner (19).

Such interactive effects between aldehydes and alky lating agentsmay contribute to the mutagenic and carcinogenic effects ofcomplex mixtures such as tobacco smoke.

One of the earliest responses to TPA is an increase in pros-taglandin synthesis (8). Although HPO increase ARA and CHrelease into the culture medium, we do not have sufficientevidence to say whether this resulted from activation of associated enzymes or from nonspecific effects in cell membranes. Thedecreased release of ARA and CH observed after ACT and FMDmay also have resulted from their interaction with membraneprotein or phospholipid.

In summary, we have characterized some of the acute effectsof FMD, HPO, BPO, or ACT on NHBE cells. Prominent findingsare induction of CLE formation without a squamous morphologychange and induction of SSB and DPC. Because these compounds do not induce a characteristic squamous morphologicalchange, require higher concentrations for effect, and have inconsistent effects on ARA release, we conclude that their mechanisms of action involve pathways different from those invokedby the phorbol ester tumor promoters.

REFERENCES

1. Bauxman, M. M., and Wuepper, K. K. Keratin cross-linking and epidermaltransglutaminase. J. Invest. Dermatol., 65:107-112,1975.

2. Bellomo, T. H., Jewell, S. A., and Orrenius, S. Regulation of intercellularcalcium compartmentation: studies with isolated hepatocytes and f-butyl hy-droperoxide. Proc. Nati. Acad. Sci. USA, 79: 6842-6846, 1982.

3. Bellomo, G., Mirabetli, F., Richelmi, P., and Orrenius, S. Critical rote of sulfhydrylgroupfs) in ATP-dependent Ca2* sequestration by the plasma membrane

fraction from rat liver. FEBS Lett., 763:136-139,1983.4. Bellomo, G., Thor, H., and Orrenius, S. Increase in cytosolic Ca2* concentration

during (-butyl hydroperoxide metabolism by isolated hepatocytes involvesNADPH oxidation and mobilization of intracellular Caz* stores. FEBS Lett768:38-42,1984.

5. Bimboim, H. C. DNA strand breakage in human leukocytes exposed to a tumorpromoter, phorbol myristate acetate. Science (Wash. DC), 275: 1247-1249,1982.

6. Bradley, M. O., Hsu, I. C., and Harris, C. C. Relationship between chromatidexchange and mutagenicity, toxicity and DNA damage. Nature (Lond.), 282:318-320,1979.

7. Bradley, M. O., and Erikson, L. C. Comparison of the effects of hydrogenperoxide and X-ray irradiation on toxicity, mutation and DNA damage/repair inmammalian cells (V-79). Biochim. Biophys. Acta, 645:135-141,1981.

8. Bresnick, E., Munier, R., and Lambdin, M. Increase in prostaglandln synthesisin tumor promotion. Cancer Lett., 7:121-125,1979.

9. Committee on Medical and Biological Effects of Environmental Pollutants.Sources of atmospheric hydrocarbon. In: Vapor-phase Organic Pollutants, pp.

CANCER RESEARCH VOL. 45 JUNE 1985

2525

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

EFFECTS OF ALDEHYDES AND PEROXIDES ON HUMAN CELLS

4-42. Washington, DC: National Academy of Science, 1976.10. Ellem. K. A. Ö. and Gierthy, J. F. Mechanism of regulation of fibroblastic

replication. IV. An analysis of the serum dependence of cell replication basedon Michaelis-Menten kinetics. J. Cell. Physiol., 92: 381-400,1977.

11. Emerit, I., and Cerutti, P. A. Tumour promoter phorbol-12-myristate-13-acetateinduces chromosomal damage via indirect action. Nature (Lond.), 293: 144-146.1981.

12. Feron, V. J. Effects of exposure to acetaldehyde in Syrian hamsters simultaneously treated with benzo(a)pyrene or diethylnitrosamine. Prog. Exp. TumorRes., 24: 162-176,1979.

13. Fischer, S. M., Mills, G. D., and Slaga, T. J. Inhibition of mouse skin tumorpromotion by several inhibitors of arachidonic acid metabolism. Carcmogenesis(Lond.), 3: 1243-1245,1982.

14. Fornace, A. J., Jr., and Little, J. B. DNA crosslinking induced by X-ray andchemical agents. Biochim. Biophys. Acta, 477: 343-355,1977.

15. Frazelte, J. H., Abemethy, D. J., and Boreiko, C. J. Weak promotion of C3H/10P/2 cell transformation by repeated treatments with formaldehyde. CancerRes., 43: 3236-3239,1983.

16. Fusenig, N. E., and Dzarlieva. R. T. Phenotype and chromosomal alterationsin cell cultures as indicators of tumor-promoting activity. Carcinog. Compr.Surv., 7:201-216,1982.

17. Goldstein, B. D., Witz, G.. Amorouso, M., Stone, D. S., and Troll, W. Stimulationof human polymorphonuclear leukocyte Superoxide and radical production bytumor promoters. Cancer Lett., 11: 257-262,1981.

18. Grafstrom, R. C., Fornace, A. J., Jr., Autrup, H., Lechner, J. F., and Harris, C.C. Formaldehyde damage to DNA and inhibition of DNA repair in humanbronchial cells. Science (Wash. DC), 220: 216-218,1983.

19. Grafstrom, R. C., Curren, R. D., Yang, L. L., and Harris, C. C. Genotoxicity offormaldehyde in cultured human bronchial dbrobiasts Science (Wash. DC),228:89-91,1985.

20. Hirota, N., and Yokoyama, T. Enhancing effect of hydrogen peroxide uponduodenal and upper jejunal carcinogenesis in rats. Gann, 72: 811 -812,1981.

21. International Agency for Research on Cancer. IARC Monogr. Eval. Carcinog.Risk Chem. Hum.. 29: 345-389, 1982.

22. Ktetn-Szanto, A. J. P., and Slaga. T. J. Effects of peroxides on rodent skin:epidermal hyperplasia and tumor promotion. J. Invest. Dermatol., 79: 30-34,

1982.23. Kohn, K. W., Ewig, L. C., Erikson, L. C., and Zwelling, L. A. Measurement of

strand breaks by alkaline elution. In: E. C. Friedberg and P. C. Hanawalt (eds.),DNA Repair, A Laboratory Manual of Research Procedures, pp. 379-401.New York: Marcel Dekker, 1981.

24. Latarjet. R. Interaction of radiation energy with nucleic acids. Curr. Top. Radiât.Res., 8: 1-38,1972.

25. Lechner, J. F., Haugen, A., McCtendon, I. A., and Pettis, E. W. Ctonal growthof normal adult human bronchial epithelial cells in serum-free medium. In Vitro(Rockvilte), 78: 633-642,1982.

26. Lechner, J. F., Haugen, A., McClendon, I. A., and Shamsuddin, A. M. Inductionof squamous differentiation of normal human bronchial epithelial cells by smallamounts of serum. Differentiation, 25: 238-246,1984.

27. Lechner, J. F., McClendon, I. A., LaVeck, M. A., Shamsuddin, A. K. M., andHarris, C. C. Differential control by platelet factors of squamous differentiationin normal and malignant human epithelial cells. Cancer Res., 43: 5915-5921,

1983.28. Lechner, J. F., and Kaighn. M. E. Application of the principles of enzyme

kinetics to donai growth assays: an approach for delineating interactionsamong growth promoting agents. J. Cell. Physiol., 700: 519-530,1979.

29. Lechner, J. F., Stoner, G. D., Haugen, A., Willey, J. C., Trump, B. F., andHarris, C. C. In vitro human bronchial epithelial model systems for carcinogenesis studies. In: M. Webber and L. Sekely (eds.), In Vitro Models for CancerResearch. New York, CRC Press, 1984, in press.

30. Lichti, U., and Gottesman, M. Genetic evidence that a phorbol ester tumorpromoter stimulates omithine decarboxylase activity by a pathway that isindependent of cyclic AMP-dependent protein kinases in CHO cells. J. Cell.Physiol., 773: 433-439,1983.

31. Massie, H. R., Samis, H. V., and Baird, M. B. The kinetics of degradation ofDNA and RNA by H2O2.Biochim. Biophys. Acta, 272: 539-548,1972.

32. McDowell, E. M., and Trump, B. F. Histological fixative suitable for diagnosticlight and electron microscopy. Arch. Pathol. Lab. Med.. 700: 405-414,1976.

33. National Research Council. Formaldehyde and Other Aldehydes. Washington,DC: National Academy Press, 1981.

34. O'Brien, T. G. The induction of omithine decarboxylase as an early, possibly

obligatory, event in most skin carcinogenesis. Cancer Res., 36: 2644-2653,1976.

35. O'Brien, T. G., Simsiman, R. C., and Boutwell, R. K. Induction of the polyamine-

biosynthetic enzymes in mouse epidermis by tumor promoting agents. CancerRes., 35: 1662-1670,1975.

36. Rhaese, H., and Freese, E. Chemical analysis of DNA alteration. I. Baseliberation and backbone breakage of DNA and oligodeoxyadenylic acid inducedby hydrogen peroxide and hydroxylamine. Biochim. Biophys. Acta, 755: 476-490,1968.

37. Rice, R. H., and Green, H. Relation of protein synthesis and transglutaminaseactivity to formation of the cross-linked envelope during terminal differentiationof the cultured human epidemial keratinocyte. J. Cell Biol., 76:705-711,1978.

38. Sakabe, H., Matsushita, H., and Koshi, S. Cancer among benzoyl chloridemanufacturing workers. Ann. NY Acad. Sci., 277: 67-70,1976.

39. Slaga, T. J., Klein-Szanto, A. J. P., Triptett, L. L., and Yotti, L. P. Skin tumor

producing activity of benzoyl peroxide, a widely used free radical generatingcompound. Science (Wash. DC), 273:1023-1024,1981.

40. Sun, T. T., and Green, H. Differentiation of the epidemial keratinocyte in cellculture: formation of the comified envelope. Cell, 9: 511-521,1976.

41. Swenberg, J. A., Kerns, W. D., Mitchell, R. I., Gralla, E. J., and Pavkov, K. L.Induction of squamous cell carcinomas of the rat nasal cavity by inhalationexposure to formaldehyde vapor. Cancer Res., 40: 3398-3402,1980.

42. Watt, M. F., and Green, H. Involucrin synthesis is correlated with cell size inhuman epidermal cultures. J. Cell Biol., 90. 738-742,1981.

43. Willey, J. C., Salariino, A. J., Lechner, J. F., and Harris, C. C. Acute effects of^-O-tetradecanoylphorbol-IS-acetate, teleocidin, or 2,3,7,8-tetrachlorodi-benzo-p-dioxin on cultured normal human bronchial epithelial cells. Carcinogenesis (Lond.), 5: 209-215. 1984.

44. Wynder, E. L, and Hoffman. D. Tobacco and health. A social challenge. N.Engl. J. Med., 300: 894-903,1979.

45. Yuspa, S. H., Ben, T., Hennings, H., and Lichti, U. Phorbol ester tumorpromoters induce epidermal transglutaminase activity. Biochim. Biophys. Acta,97: 700-708,1980.

46. Yuspa, S. H., Lichti, U., Ben, T., Patterson, E., Hennings, H., Slaga, T. J.,Colburn. N., and Kelsey, W. Phorbol esters stimulate DNA synthesis andomithine decarboxylase activity in mouse epidermal cell cultures. Nature(Lond.), 262: 402-404, 1976.

CANCER RESEARCH VOL. 45 JUNE 1985

2526

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1985;45:2522-2526. Cancer Res   Andrew J. Saladino, James C. Willey, John F. Lechner, et al.   Epithelial CellsHydrogen Peroxide on Cultured Normal Human Bronchial Effects of Formaldehyde, Acetaldehyde, Benzoyl Peroxide, and

  Updated version

  http://cancerres.aacrjournals.org/content/45/6/2522

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  [email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/45/6/2522To request permission to re-use all or part of this article, use this link

on April 19, 2020. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from