Document 6wQKYJLK4Mqn0kmdVEwezKKR4

Effects of Vinylidene Chloride on DNA Synthesis and DNA Repair in the Rat and Mouse: A Comparative Study with Dimethylnitrosamine R. H. Reitz, P. G. Watanabe, M, J. McKenna, J. F. Quast, and P. J, Gehbinc Toxicology Research Laboratory, Health Is- Environmental Sciences, USA The Dote Chemical Company, Midland, Michigan 48640 Reprinted from Toxicology and Applied Phahmacolocy, VoI. 52, No. 3, March 15, 1980 Copyright 1980 by Academic Press, Inc. Printed in U.SA. 358 REITZ ET AL macromolecules correlates well with the tumorigemc effects observed by Maltoni ei al. (1977). Binding of VDC metabolites to intracellular macromolecules is greater in the mouse than in the rat. Furthermore, the highest level of macromolecular binding is observed in the kidneys of male mice, the site where tumors were observed by Maltoni. Since it is a currently popular theory that alkylation of DNA by electrophilic species is a critical event in the induction of neoplasia (Miller and Miller. 1966). one objective of this study was to characterize the interaction of |'4C|VDC with nucleic acids in vivo. However, alkylation of DNA to initiate the tumorigenic process is not the only factor to be considered. It is known that certain chemicals and surgical procedures, while apparently not tumorigenic themselves, act to ` promote ' the induction of tumors from other sources (Van Duuren. 1969: Dateefa/., 1976). "Promotion" can be significant even if the initial oncogenic stimulus is not known. For example. Peraino et al. (1973) suggest that phenobarbital enhances the "spontane ous" rate of tumor formation in mice, while Berenblum (1929) found that repeated freez ing of the skin was carcinogenic in mice without exposure to any chemical agent. Berenblum further reported that many types of repeated cell damage can dramatically promote an oncogenic stimulus (Berenblum, 1944). Although the mechanisms of "promotion" are not well understood, a common char acteristic of chemical promoters, partial hepatectomy, and chronic irritation is that increased cell division is present (Marx, 1978). This is reflected by an increase in the amount of normal DNA synthesis as replica tion takes place. Consequently, in addition to measuring direct interaction of VDC with DNA, we have attempted to assess the "promoter" or epigenetic activity of VDC by measuring the rates of incorporation of l3H]thymidine into the DNA of various tissues. The objectives of this work were thus to investigate the importance of both genetic and epigenetic mechanisms in the induction of tumors by VDC. This k nowledge is im portant for determining whether the observa tion of tumors in mice exposed to toxic levels of VDC has any relevance for assessing the potential hazard for humans exposed to nontoxic levels of VDC. METHODS A nutuil Male mice, CD-I strain and male rais. SpragueDawley strain were obtained from Charles River Laboratories, The animals were 18-20 (mice) or 200250 g (rats) when received, and were acclimated for at least I week before use Mutf'rniU [,JC)VDC (0,441 mCi/mmol) was synthesized by New England Nuclear This material was labeled al both C-l and C 2 and had greater than 99radio chemical purity by gas chromatographic analysis I'HJThymidine (C-6 labeling. TdR) was obtained as a stock item from New England Nuclear with a specific activity of approximately 20Ci/mmol. Nonradioactive VDC (redistilled. 99 95^ pure with residual inhibitor content 1-5 ppm monomethylether of hydroquinone) was obtained from the Saran and Converted Products Research Laboratory. 1603 Building. Dow Chemical Company. Midland. Michigan. Dimethylnitrosamme (Cold Label Grade! was obtained from Aldnch Chemi cal Company. All other chemicals were of reagent grade from commercial suppliers. Enzymes used in the purification of DNA (chymotrypsin, trypsin, nbonuclease A, and deoxyribonuclease (DN-100)1 were purchased from Sigma Chemical Company Expttxitre\ Animals were exposed to VDC vapors under dynamic conditions with either a single pass system or a re circulating system, in a 30-liter glass inhalation chamber, (neither case, the concentration of VDC was measured by periodically sampling the chamber atmosphere and determining the v DC concentration by gas chromatog raphy. One-milliliter samples of the chamber atmos phere were chromatographed on a 3-ft column of Porapak QS (80/100 meshl at I50C. Quantitation was by peak height with a hydrogen Dame ionization detector. In a single pass system, total flow through the chamber was - 10 liters/min and the VDC concentra- SL 100832 \ l< I )| < K , \ \\|) || |) l'|f \ KM - 170 i I SSt> I Effects of Vinylidene Chloride on DNA Synthesis and DNA Repair in the Rat and Mouse' A Comparative Study with Dimethylnitrosamine1 R. H. Rn [/. P G. VV v i \s \bi , M J. Me Kt-ss \ . .1 F Qi \si, vmi P. J (it hkisc, l <'\K Ni hk h I iiht>f i/i. / \ fl< alfh X I n \u >,, ntn! S < t< n< . v < S A !"1 /*'" < >u HIM If/ ( <>>pnn\ Mnllilfhl \Jh /ML'iin AS'fSJU . i i/ \/<m i /) J'-r /"'C Mt ( ( pi ( J ( )< / Wm - D C "W hlfeus of Vim lulone ( hloridc on D\ A Synthesis diuf DN \ Kenuu in the K.u .md Mouse \ C ompanttive Smdv w uh DtmelhvInilrosunune Rfii/ R H Wvimsvhi P (j MiKt's'sv M J.Qivsi.J f wr fri mtiM,; P ! < I9k(h \;'pi /'Iiumimi, n/ 52. ^57 m f" xposure !o v tnv hdeno s hi on tic < V fX } .tpor hjs been r ppor led to induce tumor-, m in tee but oils .ire apparently insensitive to this effect of V!)( I his species difference has been sort elated w ith the e renter capacity of mice to activate VfX to <( te<u.tive electrophile wh^h can react with macromolecules To inere.ise our understanding of the molecular eventc associated with this speeies difference see have investigated the potential of \ Dl to cause DNA alkylation. DN A repair and DN \ rephcanon in the livei and kidnevs of i.its and mice lor comparative purposes the potent carcinogen dimeihv fnitrosamine iDMNi was also studied Male Sprague - Dave ley rats and CD-1 mice were esposed to 10 and ppm VtX for b hr DNA alkvlalion after ^0 ppm |llC']VDC was minimal in fiver and kidnev of both rats and mice tone or two orders of magnitude less than reported for |)MN tn ratsi Similarl v ON A repair m the kidnev of mice exposed to *0 ppm V DC was onh < higher lhan control vtdues while DN A repair m the liver of mice iniected with 20 mg kg DMN was elevated h57/; However, tissue damage and increased DN \ replication i2> tofdi were seen in the kidnevs of mue exposed to s() and 10 ppm VDC (. omparahle effects were no! seen m the liver of mice exposed lo VDC' i M) or 10 ppmi or in fhe liver or kidnevs of iats exposed to 10 ppm VDC fhus an important distinction between DMN and VDC has been demonstrated Tumongentc doses of DMN produced relatively litife tissue damage, but were associated with a hiuh degree of DNA alkvtaiion and DNA repau svnthesis In contrast exposure lo tumorigenic Joses of VDC resulted m massive tissue damage but induced minimal DNA alkvlalion or DNA repair svnthesis This suggests that the tumors observed m mice exposed to VDC arise pnmarilv through effects of the chemical on nongenetic components of the cells Consequent Is protection of humans I mm lev eK of V DC sufficient io cause tissue damage should also serve to preclude any carcinogenic activitv of V DC Vinylidene chloride (I. I-dichloroethylene. VDC) is an intermediate in the synthesis of many commercially important plastics. Al though carcinogenicity of this material has not been observed in several long-term studies with rats (Viola and Caputo. 1977, Rampy cl til., 1977, Maltom a a!.. 1977). This study was funded by the companies supporting the vmvhdene chloride projects being administered bv the Manufacturing Chemists \ssooalion, Wash* mgton D C VDC-related tumors have been reported in mice (Maltom cl ul . 1977). Maltom reported that male mice were fare more sensitive to VDC than females, that the target organ in male mice was the kidney, and that the tumorigemcity of VDC was associated with significant injury to kidney tissue in male mice (Maltom ct ul.. 1977). Studies by McKenna a ul (1977) have shown that the metabolism of VDC to electrophilic species which bind to cellular (HMi.imux 80oi(i"7-u$(i: non (.opvngbi < WW) bv 1`fess Itu Ml righi' i>! rtfprodiii"on in anv form r^'ffveii 100833 sh 360 RhlT/, tr M I 'MliallOtl of \ll( l(,( ijy, DNA. RNA and protem were quantitated bv the methods of Burton I 1956). Brown I 1946), and I uwry d ul (19? 11, respectively DNA was isol.ited by a modification of (he method ol Marmur il96h tissues were rapidly excised and frozen on dry we I he tro/en tissues were then bomogem/ed mOW mIDIUIOimi N.t( 1 ipH K Oi to give a final concentration of' 10'; (w vi T'he cells were then immediately Iwd by the addition of 0 I vol ot 22' > sodium dodecvl sulfate and mixed with 0 2 vol ol 5 m NaCIO) I he miMure was healed to 60 C lor 10 min. cooled, and extracted with I 0 vol ol 4'/ isoamvl alcohol tn chloroform (l hr shaking at 20-25 Cl After extraction, the aqueous laver was carefully removed DNA was spooled on a glass lod after addition of 2 vol cold 95*'' ethanol If the DN A was from a | `H |thymidme injected ani mal. it was blotted dt\ and then incubated in a minimal amount ot DN A sc ill ! my. ml enzvme in 0 05 m phos phate - 0 01 vi viy(. I... pH 7,0) at 37 C until it had dis solved Aliquots ot this solution (hvdrolyzed DNAi were counted directly or analyzed by the diphenvlamwc reaction as described by Burton t 1956k DNA from ammals exposed to |"C|VDC was sub jected to further purification After spooling the DN A was redissolved m I ml ofdislilled water 3 ml of original homogenate (gentle agitation overnight at 20-25 Li After dissolution 0 I vol of crystalline o-amylase <1 mg/ml dissolved in 0 I \i PC),. pH 7) were added and the solution was incubated for 30 mm at 77 C" Next an additional 0 I vol of a solution of trvpsin 10 5 mg, mil and chy motry psin (0 * mg ml) in 0 I \i P()t (pH 7) was added and incubation was continued for 90 mm. These enzymatic digestions degrade residual glycogen and protein in the DNA preparation After meuhation, 0 2 vol of ** m NaCIO* was added and the nucleic acids were spooled as before by the addition ol 2 vol of 95' < ethanol After redissolving in distilled water, DNA and RNA were separated as follows 0 I vol of nbonuclease A (0 5 mg ml in 0 l m PO,0 01 M curate. pH 7) was added and incubated at 37C for 2 hr DNA was separated from the hydrolyzed RNA by precipitation with 0 25 vol of 25rr trichloroacetic acid (TCA) and collected on a Millipore tiller. For assay, the DNA precipitate was dissolved in a minimal volume of 0 I mg/ml DNAse m 0 05 m PO* (pH 7 0) also containing 0 01 m MgCl- WhentheDNA was completely hydrolyzed, aliquots were sampled lor scintillation counting or colorimetric analysis IfistofuiihoiiwH ul 4 s w*v wticnf Samples ol kidney and liver were removed at necropsy and fixed m buffered I(P<' formalin, The tissues were process:11 bv louime histologic piOcCduies Sections (5-6 4m' wefv stained with hemaloxv line and cosine and examined bv light rructoscOpv Si iHtllluli- ''1 < ' tinlfnlc Samples o| up to 2 ' ml weie counted in Is ml AC S i A me i sham i on a \ uc leaf ( hk ago sc ml illation counter (Maik II or Mai k III i with quench collection S t,! \ mil\ w' / li\\ Specific t .ulio.ic 11 v i ties (dpm mg DNAi weie calculated toi each tissue ot each animal [ uch treaimenl iismic group wa- fhen unalv/ed toi a significant nic icase ov ei the appiopi iaie control by use of Studen! s/ u-si Results wete considered significant at a f` v lane 0 O' // \"( \\IH ,ilk\l,iitJ l*\ \ In orJer to obtain sut tic item mateiial lot anaksis tissues from live is* seven animals weie pooled toi sleiei mmation of the speulic ludioadiv uv ol l)N \ Nc\l ihe logarithm s>t the specific i.idio.tctiv nv idpm mg DN 4 > was subjected to linear regiessnm analysis veisus tune (hi after the let mmation of the 6 hi exposure is* S DC i III D \ \ Rti'un Nmeiv five peicent confidence intervals on the csi rectum factor were calculated alter i|i v uitng indiv idtial treatment v aloes bv the mean ol the appropriate wontiol group Specific radioactivities ot the DNA ftom individual animals m ihe subgroup re ceiving! H |thv midme and hy drox vmea alter treatment are divided bv the correction factoi and compared to individual specific radioactivities m the corresponding csmtrol subgroup using one-sided f test Increases ;ire considered significant at the /> 0 O' level i\ l)\A i>u`luhrU d n uh ] 7/j///\wiJoic Data from control and VDC -'0 ppm exposed groups at 0. 48. and I 20 hi postexposure were subjected to a two-way. completely random analy sis of variance with/ - 0 05 chosen .is the level of significance RESULTS GcnetU hJteci\ The levels of radioactivity which were incorporated into DN A purified from the tis sues of animals exposed to |4C]VDC are summarized in Table I. VDC alkylation of DNA was very low in kidney and liver of both rats and mice. The highest level of alkylation was observed in the kidneys of mice exposed to 50 ppm VDC and cor- respi nude Af VDC lame the [' expo corn, the l to l( tiv If w hil. frorr DN Kill! R\i^ Ti si) PI Ki Li 10 p K l.j 10 p I K I I 10 r at th medi as m from (10 re$i exc anir the to 4 onb sv both genetic the induction wledge is lmr the observato toxic levels assessing the .posed to non- ; rats, Spraguei Charles River -0 (mice) or 200e acclimated for . synthesized by al was labeled at than 9*Kr radiographic analysis was obtained as a jar with a specific 'I Nonradioacuve residua) inhibitor of hvdroquinone) inverted Products g, Dow Chemical neihylmtrosamme im Aldrich Cherm, were of reagent of DNA tchymodeoxynbonuclease Sigma Chemical sots under dynamic >s system or a renhalation chamber /DC was measured er atmosphere and by gas chromatogte chamber atmos( column of Porapak ution was by peak .ltion detector flow through the le VDC concentra ni\ \ i\ t> in rac i ions D\ \ ot Atm on a tion was maintained bv mcttuinc cork enir.ued ^IK v.tpoi 11000- MHHJ ppmi trv>n> a "Cp.iMic -.upplv Kig The supply hag wav prep.ued by passing 100 litci*' ol air t measured with a t) f M-115 Singer gas mereri th/oueh a U tube eoniaining 0 4 to 2 0 ml ol X IK so that the V DC ev .ipor.ued and mi xed thoi uuahly w ith (he an I-or the recirculating chamber exposures. air was Circulated ihiough Jhc w stem at TO - 0) liters mm w ith a 1 homas Model 90?C AlkTI'l; pump A,iter and ( 0_, were remov ed ti om the sy stem by L)i icrite and Ascantc traps ( ( lb of eachi in senes with the pump O was metered into the dosed sy stem to replace the C() using a Manow atch uonsi.mi pi ess u re v abe i l'R ( heltenham Pa) |''( |VD( was mieued into ihe sv stem b\ lemos mg 10 mi poitions of the headspace from a scaled septum via) I' mt s apacits, containing - - ^ ml ot < |"C|V[)( m xylene Betore removing the headspace, the septum vial was momentarily warmed to 60 ( hy immersion m a hot water bath Because ot the large differences in the sapor pressure ot \ IK and xvlene the amount ot wlene transferred to the chamber by this procedure is Jess than 2' i ot the amount ot \ DC added All inhalation exposures were 6 hr in duration and were initiated between 9xm and iZnoon Both control and exposed animals were deprived oflood and water during the exposure otherwise these were available ad Uhnwn D \ 4 S \ ntht \i\ ` ( tii I >i \ i \inn ' l>N X synthesis ssas measured 4H hr after exposuie In this procedure .1 population ot animals was di\ ided into a control and treated group Each group rece/ved an ip injection of | lH(thymidine t ^00 or I0IX) /iC i kg body wn m a volume of 0 01 ml g ot body wt I our hours after the | 'HJthymidme injec tion, the animals w ere sacrificed and DMA was isolated from kidnev and or liver as described elsewhere Increased i)N A replication was calculated hy divid ing the mean specific radioactivity ot DNA prepara (urns trom treated groups by the mean specific radio activity ot the DNA preparations from the control group It was noted that the degree of labeling of DN A with | (H [ihv rnidine was relatively constant within a matched group of animals ti e animals with a given birth date, taken from the same shipment and housed identically until use) but could vary to two- or threefold between different shipments of animals Consequently, each experiment includes a matched control group and the ration of incorporation in treated and control groups is calculated as a "replicative index A value of 1 0 or less indicates that the treatment did not stimulate DN A replication, while numbers larger than l 0 show stimulation of DNA replication, indicating regeneration following cellular injury /)\ \ Hryan DN A i epair w,is measuied i^.<wding to the follow ing piiKcduie umm.ils from one popi:\t(ion were divided into neuted i I i or control (> groups bach group was further divided into [wo subgroups fibres to si\ am m.ils subgroup) < )ne subgioup received i 'H |thvmidme it) 01 ml g body wi MM) oi 1000 MCi kg 20 Ci mmol ip) any! the other subgroup tcceived an identical injec oo n ot 1 H |thy mi dine dissol v cd m 40 mg. ml h y drox t urea ) our hours later, the animals were sacrificed by tcrviLiil dislocation and the DNA was prepared trom tissues as described elsewhere Re pa a was calculated from these groups as sug gevted by Artellini <( <ii il^Xi In this procedure hydroxyurea was administered simultaneously with | 'H |thy midine This siirpresses norma! replicative QN A synthesis but does noi inhibit DNA repair systems K leaver 1969) This allows more sensitivity tor the detection ot the relatively small amount of DNA repair synthesis In addition, a treated and control group injected with | `H(thymidine but not hydroxyurea were included in each experiment bvaluation ot the data Irom the tour groups allows calculation of the repair rally) as Billows Mi The specific radioactivities ot the DNA isolated from each tissue were calculated tdpm mg DN A> (2) Next a correction factor <CF> for the effect ol the test whemic.il on normal replicative DNA synthesis was ^akulated by dividing the mean specific radioac tivity ot the DNA in the treated group by that of the control group iM 1 malK the repair ratio iKRi was calculated as where (Tim - the mean specific radioactivity of the DN A isolated from the treated subgroup injected with I'Hjthymidine plus hydroxyurea. (Clm = the mean specific radioactivity of the DNA isolated from the control subgroup injected with ('HRhymidine plus hvdroxyurea and CF is the correction factor calculated in 12) Repair synthesis of DNA is maximal within a few hours after treatment, when absorption, distribution, and (in the case of proearcmogensi metabolic activa tion have taken place Consequently, repair measure ments were earned out 4 hr after ip injection of DMN. In the case of VDC exposure, animals receive the material throughout the 6-hr exposure chosen to ap proximate occupational exposures Consequently, metabolic activation of material inhaled early m the exposure has proceeded for nearly 6 hr by the termina tion of (he exposure Therefore repair measurements were begun immediately after removing the animals from the chamber, & I 362 REITZ ET AL TABLE 2 Specific Radioactivity of ( 1H]Thymidinf.-Labh ed DNA in Mice with and without Exposure to 50 ppm Unlabelld VDC (6 hr)',ft Specific radioactivity of DNA (dpm/mg DNA, mean i SE) Tissue Time post-VDC exposure (hr) 0 OO O Kidney Control VDC (50 ppm)' Liver Control VDC (50 ppm) 11 6 t 1.8 (a =4) 16 4 i 0 99 13.8 ;46(/i = 4) 17 4 i 3.4 is : i i.o 17 9 - 13 17 3 -- 1.9 15 3*32 i:,i r : i 15 5 = 0 72 18 7 - 7.6 11.4 * 2 4 " Animals were injected with | 'H|thymidine 3 days prior to the start of the experiment in order to label their DNA. Then half the animals were exposed to 50 ppm VDC sapor (nonradioactivel for 6 hr. while the remaining animals were held as a control Then the animals were sacrificed at the indicated times and the specific radioactivity of the DNA in various tissues was determined as outlined under Methods. Unless otherwise indicated, n - 5 in all groups. " Data were analyzed by a two-way analysis of variance, completely randomized design. There were no significant time effects and no significant interactions between time and treatment, but a significant ip < 0.01) treatment effect was found in the labeling of kidney DNA ' Significantly different from control, p < 0.01. dpm/mg DNA immediately following ex posure had fallen to 4.3 dpm/mg 96 hr later.) However, calculation of the kinetic param eters following following the 10 ppm exposure was not possible because of insufficient data. The loss of llC from the DNA preparations of mice exposed to 50 ppm VDC appeared to be biphasic. An initial rapid phase was noted during the first 8 hr following exposure. with a slower, log-linear phase continuing for at least 192 hr. (Apparent half-lives were calculated from the second phase.) Another experiment was carried out to establish that cell necrosis produced by VDC had not contributed to the previously ob served loss of radioactivity from the DNA of animals exposed to [,4C]VDC (Fig. I). In this additional experiment DNA was labeled 48 hr prior to treatment by injection of [3H]thymidine. The specific radioactivity of DNA isolated from the kidneys of mice previously labeled with l3H)thymidine was relatively constant, either with or without exposure to 50 ppm VDC (Table 2). The specific radioactivity of DNA from the livers of these animals also appeared fairly constant (Table 2), but a higher degree of variation in this tissue made it difficult to assess this point precisely. A two-way analy sis of variance indicated no significant time effects or interactions, but a small treatment effect was noted in the kidney ( -- 20% in crease in specific radioactivity, Table 2). DNA repair was measured directly in ani mals exposed to VDC (50 and 10 ppm) or injected with DMN (20, 10, and 3 mg/kg, ip). DMN treatment caused a dramatic in crease in the level of hydroxyurea-resistant pH]thymidine incorporation. The relative increase in repair synthesis caused by DMN, after correction for the effects of DMN or nonrepair synthesis as outlined under Meth ods. was more than sevenfold, and this in crease was dose related (Table 3). In contrast, a much smaller increase in the repair ratio (1.00 -- 1.38) occurred after exposure of mice to VDC. The only statistically signifi cant increase caused by VDC was that oc- curnt to 50 altere 50 pp mice Dose 1 Liv l.iv SL 100836 it pi ocedui L-s Sections cmalou line and cosine opv c v ounled in I ^ ml \C S ugo scintillation umn(i.,i lit It tor reclion i adui.K 11 v ines i dpm mg h iisMie ot each animal s.i"h then analyzed tor a piopnate control by use a consider'd significant t In order to obtain s (issues tiom tise to ,'i deieimmahon ot (he Next the logarithm ot mu ON AI was subjected eimis nme <hi alter the ue to VDO use percent confidence nr were calculated alter dues bv the mean ot the acvific radioactivities ot nals in the subgroup re i ox\ uiea after treatment i.utor and compared to ies m the corresponding Jed ! lest Increases .ue 0 05 level // \ih\ innlim Data ti nm \posed groups at 0. 4X subjected to a two-wav i v unance with /> = 0 0* ante TS Ltivity which were purified from the Us ed to [4C|VDC are VDC alkylation of kidney and liver of he highest level of d in the kidneys of pm VDC and cor / \ \ l \ () I \ f K R A ( T f () N S () I VIM W I I H DN A responded to only 30 alkylations per million nucleotide residues After exposure to either 50 or 10 ppm VDC. the DNA from the liver of mice con tained 5- to 10-fold less radioactivity than the DN A from the kidneys (Table 1). In rats exposed to 10 ppm VDC, the DNA of kidney contained about twofold greater activity than the DNA ot liver Compared to mice exposed to 10 ppm VDC. about sixfold less radioac tivity was found in the DNA of rat kidnev s, while similar amounts were found in the DNA from the liver of both species (Table I) yu'i '/( , 361 I \BLh I DN >\ D\M\(H PKnlM < M> KV VlN'rlllMM- ( H I <'K11) I I V DC I VM) DlMlimiMT R()S W1I M (DMSl is Rais \m) Mki 1 I reatmenL tissue dpm mg DNA A Ikv la lions nucleotide t - 10* ) *0 ppm VDC i mouse) Kidnev 1 iver XX la - h 1X l a - | ) to 61 10 ppm VDC (mouse) Kidney l iver 34 1 3 4 <h - 2) 2 XI) * () Ola - 2) tI 0 94 10 ppm VDC <rat) Kidnev l.i v er 10 mg kg DMN (rail L.i\ er 5 9 : 1 9 1 m -- 3) 2 6:19 Iff = 61 :o 0 A0(K) -4<MKC ' Animals were exposed to lMC|VDC vapor tor b hr at the indicated concentrations Then they are im mediately sacrificed for analysis Data are expressed as mean specific radioactivity - SD of DNA isolated from the pooled tissues of five (50 ppm mice), seven (10 ppm mice), or two animals (10 ppm rats) The results are the average of multiple determination, except for the data of ^0 ppm VDC mouse These animals were part of a larger experiment to determine the nme course of DNA repair in animals exposed to *0 ppm VDC and this particular time point was only determined once '' Data from Pegg and Hui (1978) I u, I Time course for radioadiv itv in the DNA ot CD-I mice exposed to r`l|kl)C Animals were exposed to 50 ppm |'`C|VDC` tor b hr \t the indicated nmes the animals were sacrificed and DN A was isolated Irom the pooled tissues ol tiv e mice nme point Hyjrolv/ed DNA (5-20 mg) was counted until at least I O'* disintegrations had been accumulated In each case the counting rate was at least twice background Hall-lives were calculated from linear regression anal\sis. and were 83 (kidnev i and 9| hr (liver) Halflives were calculated from the terminal log-linear phase ot the curves, One point was eliminated from the regression analysis tor liver l)N \ (24-hr point) alter it was found that the observed point differed signiheantly from the predicted value tor that time </ 0 015, one-sided i test) The coefficients for the cal culated regression line, logtdpm mg DNA) - a b mine m hr) were n - 1 72. h - o (MHW n 0 00034? ikidneyoiindtf = I ]6.b - 0 00329 - o 0005bI diver) Correlation coefficients tor the linear regressions are o 9967 (kidney) and 0.9928 (liven ' Point not included in regression analysis The radioactivity in the DNA from the liver and kidney of mice exposed to |,4C|VDC is rapidly eliminated (Kig. I). The ap parent half-lives were 83 and 91 hr for kidney and liver, respectively (50 ppm VDC ex posure). Radioactivity in the DNA from the kidney of mice exposed to 10 ppm VDC appeared to be eliminated more rapidly 130.2 SL 100837 t 364 REITZ ET AL. TABLE 4 DNA Synthesis in Tissues of Vinylioene Chlo ride (VDC) and Dimethylnitrosamine (DMN) Exposed Rats and Mice (Treated/Control)0 Dose/tissue Mean spec act = SE Ratio VDC (50 ppm) (mice) Kidney 272 * 15.2 (/i - 4) It 0 + 2.63 (n = 2) 24 7" Liver VDC (10 ppm) (mice) Kidney Liver VDC (10 ppml (rats) Kidney Liver DMN (20 mg/kg) (mice) Liver DMN (10 mg/kg) (mice) Liver DMN (3 mg/kg) (mice) Liver 26.9 5 40 (n = 4) 11.0 + 3 0(/i = 2) 95.0 15 2 (a = 5) 12.3 2 41 (n = 5) 17 6 + 2.96 (n = 6) 14 7 + 1.95 (n - 5) 23 8 * 3.7 (n = 3) 10 8 + 3.8 <n = 3) 16 3 - 0.87 <n = 3) 18.6 : 2.3 (n = 3) (Died before 48 hr) 94.0 + 30.1 (n = 3) 47 8 + 17.6 in = 4) 54.5 + 11.0 in - 3) 47 8 + 17.6 in = 4) 2.45 7 72* 1,20 2 20* 0.88 1.97 1.14 " DNA synthesis was estimated by determining the specific radioactivity of DNA following |3H]thymidine (|'H]TdR injection. (3H]TdR was injected 48 hr after the treatment suspected of causing cytotoxicity. A positive response is indicated by an increased rate of incorporation of []H)TdR into DNA relative to a control group, resulting in a ratio greater than 1.0. * Ratio significantly greater than one ip < 0.03, t-iest). procedure differs from that employed to measure DNA repair in two important re spects: (1) Hydroxyurea is not used to suppress normal DNA replication. (2) [3H]Thymidine is injected approxi mately 48 hr after the event suspected of causing tissue damage. The results of these experiments are sum marized in Table 4. In each case, the in corporation of 3H into DNA in the treated group is compared to the incorporation of an untreated control group. Exposure to 50 ppm VDC caused about a 25-fold increase in incorporation of l3H]thymidine into DNA of mouse kidneys, while exposure to 10 ppm VDC produced about an 8-fold increase in [3H]thymidine incorporation in this tissue (Table 4). These effects probably reflect in creased levels of DNA replication rather than changes in the DNA precursor pool sizes, since a large number of mitotic figures were seen in treated, but not control, tissues during microscopic examination (Table 5). Effects on DNA replication in the mouse liver were much smaller: 2.4 and 1.2 times control at 50 and 10 ppm VDC, respectively (Table 4). DMN treatment also produced only a small increase in replication of DNA in the livers of treated mice: 1.1 and 2.0 times control at 3 and 10 mg/kg, respectively. Rats exposed to 10 ppm VDC exhibited, much smaller effects on DNA replication than mice exposed to the same concentra tion. In the rat kidney DNA replication was only elevated 2.2-fold while DNA replica tion in the liver was slightly decreased. DISCUSSION One of the most popular theories ofchem ical carcinogenesis is that alkylation of DNA by reactive molecules induces somatic mutations which permanently transform target cells into malignant clones. This theory is supported by the finding that many potent carcinogens have been shown to cause covalent alkylation of various sites in the DNA molecule. For example, Peggand Hui (1978) found that guanines in the DNA isolated from the liver of rats injected with DMN (10 mg/kg ip) were methylated to the extent of 0.3-0.4% (3000-4000 mol of vc VC DV D5 SL 100838 . I T" MOVJ T * SE) 120 s s * o 72 87:76 114:24 in order to label >r 6 hr. while the ed times and the Methods Unless n There were no ificant (p < 0.01) vlA from the ppeared fairly her degree of it difficult to wo-way analyignificant time nail treatment ey (-20% iny, Table 2). iirectly in aniid 10 ppm) or and 3 mg/kg, i dramatic inurea-resistant The relative lsed by DMN, is of DMN or d under MethJ, and this in3). In contrast, le repair ratio exposure of tically signifi- was that oc- is V / V O INTERACriONS OF VDC WITH ON A 363 curring in the kidneys of the mice exposed to 50 ppm. DNA repair was not significantly altered in the livers of the mice exposed to 50 ppm VDC, or in the kidneys or livers of mice exposed to 10 ppm VDC (Table 3). Epigenetic Effeas A second procedure which measures pri marily DNA replication was used to evaluate nongenetic effects of VDC exposure. This TABLE 3 DNA Repair is Tissues of Mice Exposed ro Dimethylniprosxmine (DMN) or Vin m idene Chloride (VDC). Rxrio of Treated Animms in Controls" Dose/tissue (A) I'HlTdR only (Mean spec act r SE (treated/controll (B) Correction factor (95^f confidence limit) (C) |'H|TdR - HU (Mean spec act SE) (treated/control) (Dl Repair ratio [(C) divided by (B) | (observed/ expected. 95r/r confidence limit) V DC-50 Kidney Liver VDC-10 Kidney Liver DMN 120 mg/kg) Liver DMN (10 mg/kg) Liver DMN (3 mg/kg) Liver 5 .30 -- 1 15 (it = 4>t, 9 45 2.28 (n = 3) 9 69 * 1 50 In = 4) 12.9 1,63 in - 4) 14 7 = 1.20 in = 6) 14 6 1.86 in = 6) 17 2 2.36 (n = 6) 15 6 i 1 28 (it = 6) 34 4 1.03 (n - 3),,, 80 5 1.92 (n = 3) 27.0 2 94 (n ^ 4>,,, 41,6 14 5 (n - 4) 19.0 1.41 (it = 25,0 4.93 (it = 6) l--"l o 0 561 0.285'' 0 751 0.275 I 01 0 164 0 427 : 0 038" 0,649 0 166" 0,760 0 114" 1 74 0,0488 (n = 4) 2 25 0 0917 (n - 4) 16.0 2 13 (n = 4) 18.4 0 434 (it = 4) 2 67 0.197 (it = 3) 2 27 0 640 (it =4) 2 16 0,195 (it = 3) 2 57 0 378 (n = 4) 44 7 4 51 (tt = 3) 14.2 2 64 (it = 3) 15 5 1 95 (it = 4) , 7 85 1,28 (it = 3) 9.57 t 2 32 (ft = 3) 7.85 1 28 (n = 3) 1 38 0 090" 116 0 363 116 0 255 0 764 0 222 7.37 2.16* 3,04 0.896s 1.60 1 14 " Animals were injected with (3H]thymidine (l3H]TdR) or pH|TdR plus hydroxyurea (HU). HU is given at the same time as [3H]TdR in order to block most of the replicative DNA synthesis. The times of [5HJTdR or [3H]TdR + HU injection were (a) immediately following a 6-hr inhalation exposure to VDC, or (b) 4 hr after ip administration of DMN The amount of DNA repair is reflected in column (D) where the ' repair ratio," calculated as outlined under Methods, is presented. A value of I 0 in this column indicates little or no repair is present, while any increase in this value above 1.0 is taken as indication that prior DNA damage has occurred. The repair ratio is the ratio of the observed hydroxyurea-resistant TdR incorporation to that which would be expected from consideration of corrected control values. It is calculated as (C) divided by (B) as outlined under Methods ' Ratio is significantly different than 1,0. i test with p < 0 05. ' Animals received 1000 jtCt/kg I 'H|thymidine instead of the 500 jiCi/kg used in the rest of the experiment. SL 100839 366 REITZ ET AL. that the mutagenic effects of this chemical will be much less than those produced by a potent in vivo alkylator such as DMN. Since the determination of radioactivity in the DNA preparations of these animals was used to indicate the degree of genetic damage, it is important to eliminate the possibility that this radioactivity could arise from sources other than DNA alkylation. Two such sources might be: (1) contamina tion of the DNA preparation with other macromolecules which are radioactive (e.g., protein), and (2) breakdown of [MC]VDC by metabolism with subsequent incorpora tion of radioactivity into DNA from the C-l pool (Gillette and Pohl, 1977). Contamination of DNA To eliminate contamination as a source of radioactivity, the DNA preparation was extensively purified. In addition to the basic procedure of Marmur (1961), the DNA was treated with purified enzymes which specifi cally degraded protein (trypsin and chymotrypsin treatment), glycogen (a-amylase treatment), and ribonucleic acid (ribonuclease A treatment). Analysis of the final prepa ration of DNA indicated that it contained less than 0.6% protein and less than 2% RNA by weight. The high purity of the DNA preparations makes it unlikely that contami nation is responsible for the radioactivity observed. Incorporation of C-l Fragments If the majority of the radioactivity in the DNA prepared from animals exposed to [MC]VDC had arisen from incorporation of radioactive C-l fragments, then it should appear in normal (i.e., nonalkyiated) DNA bases. Consequently, this radioactivity should be very persistent. (Gross et al. (1969) report a /V4 of >30 days for liver DNA). However, if the radioactivity in the DNA has arisen by alkylation, DNA repair systems should cause it to be lost much more rapidly. In order to investigate this point, DNA was isolated from |'4C]VDC exposed animals at various times after exposure. As shown in Fig. 1, this radioactivity was rapidly eliminated, with half-lives of less than 100 hr. Cell necrosis could not be responsible for this loss since it was shown in another experiment that exposure to the same concentration of VDC (50 ppm) did not cause the turnover of normal DNA pre viously labeled with (:iHjthymidine (Table 2). Since 70-80% of the radioactivity was lost in the time period studied (192 hr), it appears that most of the radioactivity found in DNA after exposure to lHC]VDC is recognized as "damage" by the DNA repair systems, and consequently represents direct alkylation rather than incorporation of C-l fragments. The functioning of DNA repair mechanisms in response to this small amount of damage is a significant observation, because these repair mechanisms are thought to play an important role in protecting or ganisms from small amounts of genetic damage. In order to carry out a direct comparison of the genetic effects of DMN and VDC in the mouse, a second experimental pro cedure for assessing DNA repair in vivo (as an indicator of preceding DNA damage in vivo) was developed. [3H]thymidine was used to measure in vivo DNA synthesis in the presence of hydroxyurea (400 mg/kg) (see Methods). DMN treatment caused a dramatic in crease in the level of HU-resistant [*H]thymidine incorporation (Table 3). The ratio of observed/expected incorporation (Repair Ratio, Column D in Table 3) was elevated more than sevenfold at the highest dose of DMN (20 mg/kg) and declined in a doserelated fashion. In contrast, VDC exposure caused only a slight increase in this ratio (1.00 - 1.38) in the kidneys of mice exposed to 50 ppm. The "repair" ratio in the livers of these animals, or the livers or kidneys of mice exposed to 10 ppm of VDC, was not \ signific repair i too low depend order t induce< Sino implies tion to tency c that its genic e The fail effects suggest be opei Thet toxic) tumorii et al. clear-c degree cinogei species Ther peated to caus rate of obviou replica: signific regenei study such r fidelity for spo Furthei arising cosmic as "'em that the some a the orglevels regenet found hanced events SL 100840 njected approxisnt suspected of riments are sumich case, the in(A in the treated incorporation of ). Exposure to 50 25-fold increase midine into DNA posure to 10 ppm Mold increase in on in this tissue obably reflect ineplication rather \ precursor pool of mitotic figures it control, tissues nation (Table 5). lion in the mouse 2.4 and 1.2 times 'DC, respectively nt also produced plication of DNA nice: 1.1 and 2.0 ^kg, respectively, n VDC exhibited DNA replication same concentraA replication was iile DNA replicatly decreased. )N theories ofchemilkyiation of DNA induces somatic nently transform lones. This theory ; that many potent shown to cause nous sites in the pie, Pegg and Hui nes in the DNA rats injected with methylated to the >00- 4000 mol of i\ vnu intf.ractions ok vdc with dna 365 TABl.F. 5 $LMMARY OF HlSTOPATHOLOOS FlNDlNOS IN TlSSLl s I ROM MlCF TrEaTFO WITH V l N X I iDt'NF C M LORI Ob (V[)C) OR DlMT.THY I.MI ROSAMlNF (OMN)" Exposure Postexposure (hr) Histologic findings Kidneys Liver VDC t50 ppm). 6 hr VDC < 10 ppm) 6 hr DMN ( 10 mg;kg ip) DMN (\ mg/kg ip) 0 Toxic nephrosis 8 Progressing nephrosis 24 Progressing nephrosis 48 Increased mitotic figures 06 Regeneration apparent 102 Regeneration continuing Slight centnlobular swelling No effect 0 Slight dilation, swelling % Nephrosis-variable O-ZO'T affected 4 Centnlobular swelling Hyaline degeneration 52 Accentuated lobular pattern. necrosis minimal or absent 4 Centnlobular swelling 52 No effect '* Three to six animals were given the indicated treatment and then sacrificed at the appropriate time. I issue slices were prepared by standard histochemical procedures methylated base/10'' mol of guanine). In order to see whether VDC inhalation pro duced similar effects, groups of rats and mice were exposed to [UC]VDC vapor, and the DNA from various tissues of these animals was isolated and purified. The most striking observation in these studies was the extremely low degree of alkylation detected following |,4C]VDC exposure. For direct comparison with the data of Pegg and Hui (1978), the degree of alkylation of DNA is calculated from the disintegrations per minute observed per milligram DNA and the specific radioactivity of the VDC. Assuming the average molecular weight of a nucleotide residue in DNA is 330, the maximum level of DNA alkylation by VDC was 0.003% or 30 alkylations/106 nucleotides (Table 1). This occurred in the DNA isolated from the kidneys of mice ex posed to 50 ppm of [14C]VDC. When mice were exposed to 10 ppm VDC, about onethird as many alkylations occurred in the DNA of the kidney. At a site where VDCrelated tumors were not observed (mouse liver) or in the kidney or liver of an insensi tive species (the rat), alkylation of DNA by VDC was even less. Assuming that the degree of alkylation of guanine is typical of other DNA bases, we see that DMN pro duced about two orders of magnitude more DNA binding than VDC. The doses of DMN and VDC (10 mg/kg ip, and 50 ppm inhala tion, 6 hr) are about the highest levels that can be administered to CD-I male mice without causing some delayed mortality, and are at least roughly equivalent in terms of their acute toxicity. There are many potential sites for alkyla tion of DNA by VDC, and it is almost cer tain that some of these sites will differ from others in terms of their susceptibility to re pair and/or ability to cause mispairing of bases during replication. Nevertheless, the observation that overall in vivo alkylation of DNA by VDC is very low is suggestive r 368 REITZ ET AL. results are particularly significant in view of the fact that the mice in Maltoni's study (Maltoni et al., 1977) developed treatmentrelated tumors in the kidney, but not m the liver. It is also noteworthy that in the male rat, a species which is apparently resistant to the tumorigenic effects of VDC, effects on DNA synthesis were minimal. DNA replication in the kidney of VDC-treated rats (10 ppm, 6 hr) was elevated twofold (Table 4), while VDC treatment had no de tectable effect on DNA synthesis in the liver of rats. DMN was very different than VDC in its effects on DNA replication in mice. After 10 mg/kg DMN, the DNA replication in the livers of treated mice was elevated less than twofold over controls, while in mice receiving 3 mg/kg DMN this parameter was elevated only 14% (Table 4). Since the liver is a target organ for tumor induction after chronic administration of DMN in mice (IARC, 1972), it is appropriate to compare changes induced in this tissue by DMN to changes induced in the kidneys by VDC. When this is done, it is clear that exposure of mice to levels of VDC found by Maltoni to cause kidney tumors causes a dramatic increase in DNA synthesis, while treatment of mice with the known mutagen DMN at a carcinogenic dose failed to elicit a com parable response. To verify the contention that comparable degrees of tissue damage were not pro duced by DMN and VDC, tissue samples from treated mice were taken for histopathological assessment. The results are summarized in Table 5. As reported by Maltoni et al. (1977), the kidney tissue was severely affected. VDC exposure (50 ppm) produced severe necrosis, and some necrosis was also seen after 10 ppm VDC. Liver tissue was much less sensitive to the effects of VDC. DMN (10 mg/kg) produced some histopathology in the liver, but it is note worthy that this was not a necrotic type of reaction. The kidney damage produced by 50 ppm of VDC showed progressive necrosis for the first 24 hr with regeneration and recovery beginning at 48 hours, and con tinuing throughout the 192-hr period. Treat ment related effects were still apparent in the kidney at 192 hr postexposure, but a significant recovery had occurred at this time. The data presented indicate that DMN has a strong effect on genetic material. DNA alkylation and DNA repair induced by alkyla tion with DMN can be detected at doses where there is little or no cytotoxicity. In contrast, VDC has very little potential to damage genetic material, but can cause extensive cytotoxicity with concomitent cell regeneration and DNA replication. Thus, although VDC may have little propen sity to "initiate" a tumor, it could easily serve to "promote" tumors when exposure is sufficient to cause significant tissue damage. In this respect it may resemble agents such as chloroform. These materials induce tumors in animals even though they apparently have little or no genotoxic ac tivity, provided that they are administered at levels which cause chronic tissue damage. Most long-term carcinogenesis bioassays do not distinguish between agents which induce tumors through direct effects on DNA and agents which enhance or "pro mote" a low level of spontaneous tumor formation. Although the endpoint of each process is the same (tumor formation), there are fundamental differences in the safety precautions appropriate to each case. Agents which damage DNA (genotoxic) often produce a hidden effect which is not apparent until tumors appear. In contrast, agents which produce their effects through epigenetic mechanisms (cytotoxic) should not produce tumors at levels where tissue damage is absent. Furthermore, although it is theoretically possible that a single "hit" on genetic material might be sufficient to initiate a tumor (if one ignores the role of DNA repair mechanisms), it is clear that multiple "hits" in the cytoplasm would be required to kill a cell. Consequently there is ample precedent for suggesting that the rela- tive turn should sf critical dt It has 1977) tha activate ' appears sensitivity mice met rats, and terms of morigenic metabolic apparent; sistently s more sign amount of to equival For m halogenat oxidative related to the body implies tl these matt man than and indeet roform (Rt (Butcher t Conscqi study supi effects of curring at be associa be far less dose level more, sine primarily t it does not of VDC w' will pose a Arfellini. In vivo D adtninistra fortchung St, 100842 T be lost much more stigate this point, UC]VDC exposed s after exposure, radioactivity was half-lives of less isis could not be once it was shown .at exposure to the /DC (50 ppm) did ' normal DNA preJthymidine (Table radioactivity was tudied (192 hr), it adioactivity found ; to [HC]VDC is by the DNA repair ly represents direct corporation of C-l ing of DNA repair 0 this small amount icant observation, lanisms are thought e in protecting ornounts of genetic direct comparison f DMN and VDC experimental pro\ repair in vivo (as g DNA damage in `HJthymidine was DNA synthesis in yurea (400 mg/kg) ed a dramatic inHU-resistant 13H)(Table 3). The ratio orporation (Repair >le 3) was elevated he highest dose of eclined in a doseist, VDC exposure rease in this ratio ys of mice exposed ratio in the livers ivers or kidneys of 1 of VDC, was not /V vivo interactions of WITH DNA 367 significantly elevated. Although the levels of repair observed after VDC exposure were too low to permit the evaluation of their dose dependency, they were clearly of a different order of magnitude than the DNA repair induced in the livers of DMN-mjected mice. Since the observation of DNA repair implies that some sort of previous altera tion to DNA has taken place, the low po tency of VDC in this system again suggests that its in vivo potential for producing muta genic effects is much less than that of DMN. The failure to demonstrate significant genetic effects with tumongenic doses of VDC suggested that epigenetic mechanisms might be operating. The first suggestion that epigenetic (cyto toxic) factors may be important in the tumorigemcity of VDC came from Maltoni et al. (1977) who reported: "There is a clear-cut direct relationship between the degree of toxic (tissue damage) and car cinogenic effects in the different animal species and sexes considered." There are several ways in which the re peated induction of tissue damage sufficient to cause regeneration could influence the rate of tumor expression. First, there will obviously be a much greater number of replications of DNA in a tissue where a significant number of cells are replaced by regeneration during each day of a two year study than in undamaged tissue. Even if such replication occurred with very high fidelity, there would be an increased chance for spontaneous errors and hence mutation. Furthermore, sites of damage in the DNA arising from background sources (e.g., cosmic radiation) may not be recognizable as "errors'' after replication. This suggests that the DNA repair systems might lose some of their effectiveness in protecting the organism from the consequences of low levels of genetic damage when cells are regenerating rapidly. Berman et al. (1978) found that increased division of cells en hanced their susceptibility to mutagenic events. McCormick (1979) also reported that momentarily arresting cell division to give added time for DNA repair greatly diminished the mutational activity of uv light. In this context it is noteworthy that most of the chemical and physical treat ments which act as promoters of carci- , nogenesis also stimulate DNA synthesis (Berenblum, 1944; Marx, 1978). Although it is not clear which of these mechanisms may be significant biologically, it is clear that when the doses of chemical employed in a carcinogenesis bioassay are toxic enough to stimulate cell regeneration, epigenetic factors cannot be excluded. In order to determine whether cell division might be important in the oncogenicity of VDC, an experimental procedure for meas uring DNA synthesis was developed. This procedure differs from that employed to measure DNA repair in two important respects: (1) Hydroxyurea is not used to suppress DNA replication. (2) [3H]TdR is injected approximately 48 hr after the event suspected of causing tissue damage. Since normal replicative synthesis is much more extensive than the HU-resistant re pair synthesis of DNA, and since repair synthesis declines rapidly in the first few hours following carcinogen exposure (R, H. Reitz, unpublished data), this procedure measures primarily DNA replication. In contrast to its weak genetic effects, VDC produced a dramatic change in DNA synthesis in mice (Table 4). After exposure to 50 ppm VDC for 6 hr, the DNA replica tion in the kidneys of male mice was elevated 25-fold. This effect was also seen after exposure to 10 ppm of VDC (6 hr), where DNA synthesis was elevated eightfold over controls. However, the effect of VDC on replication of the DNA in the liver of male mice was much less; at 50 ppm VDC the liver DNA synthesis was 2.4 times control, while after 10 ppm VDC the liver DNA synthesis was only 1.2 times control. These 100843 SL 370 REITZ ET AL. P. J. (1977), Pharmacokinetics of vmylidene chloride in the rat. Environ. Health Perspect 21, 99- 105. Miller, E. C., and Miller, J. a. (1966). Mechanisms of chemical carcinogenesis. Nature of proximate car cinogens and interactions with macromolecules. Pharmacol. Rev. 18, goj. Pecg. A. E., and Hui, G., (1978). Formation and subsequent removal of 0`-meihylguamne from de oxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine Biochem. J 173, 739-748. Peraino, C . Fry. R. J M., and Staffeld. E (1973). Enhancement of spontaneous hepatic tumongenesis in C3H mice by dietary phenobarbital. J. Nat. Cancer Inst. 51, 1349-1350. Rampy, L. w., Quast, J. F., Humiston, C. G., Balmer, M F, and SchweTZ, B. A, (1977). Interim results of two year toxicological studies in rats of vmylidene chloride incorporated in the drinking water or administered by repeated inhala tion. Environ Health Perspect. 21, 33-43. Reitz, R. H., Gehring, P J , and Parr, C. N. (1978). Carcinogenic nsk estimation for chloroform1 an alternative to EPA's procedures. Food Cosmet. Toxicol. 16, 511-514. Van Duuren, B. L. (1969). Tumors promoting agents in two stage carcinogenesis. Prog. Exp. Tumor Res 11, 31. Viola, P. L., and Caputo, A. (1977). Carcino genicity studies on vmylidene chloride. Environ. Health Perspect. 21, 45-47, Weiss, M., Sziegoleit, W., and Forster, W. (1977). Dependence of pharmacokinetic parameters on the body weight. Ini. J. Clin. Pharmacol. 15, 572-575, eration and s, and conmod. Treatapparent in sure, but a at this time, at DMN has erial. DNA ;d by alkylasd at doses toxicity. In potential to can cause oncomitent replication, ttle propenould easily :n exposure ;ant tissue y resemble ;e materials hough they notoxic ac- iministered ue damage, s bioassays ents which effects on e or "protous tumor int of each formation), ces in the each case, (genotoxic) hich is not n contrast, ;ts through vie) should here tissue although it ingle "hit" efficient to the role of clear that t would be itly there is lat the rela IN VIVO INTERACTIONS of VDC WITH DNA 369 tive tumorigenicity of cytotoxic agents should show an abrupt decrease below a critical dose. It has been reported (McKenna et at. 1977) that the capacity to metabohcally activate VDC to an electrophilic species appears to be directly correlated with sensitivity to toxic effects of VDC. Thus, mice metabolize VDC more readily than rats, and are more sensitive to VDC in terms of tissue damage and attendant tu morigenicity. This association between metabolic activation and toxicity is also apparent at the molecular level. Mice con sistently showed more DNA damage, and more significantly, greater elevations in the amount of DNA replication than rats exposed to equivalent doses of VDC. For many xenobiotics, including the halogenated hydrocarbons, the rate of oxidative metabolism appears to be roughly related to the body surface area rather than the body mass (Weiss et at., 1977). This implies that the metabolic activation of these materials would occur more slowly in man than in the small laboratory rodents, and indeed this appears to be true for chlo roform (Reitzei at., 1978), and vinyl chloride (Butcher et at., 1978). Consequently the data presented in this study support the hypothesis that the toxic effects of VDC in man, including those oc curring at the molecular level and though to be associated with carcinogenicity, should be far less than those observed at equivalent dose levels in laboratory animals. Further more, since VDC appears to induce tumors primarily through epigenetic mechansisms, it does not appear that exposure to levels of VDC which do not cause tissue damage will pose any significant cancer risk. REFERENCES Arfellim. G., Grilli. G . and Prodi. G (|978i In vivo DNA repair after N-methyl-N-nitrosourea administranon lo rats of different ages 7. Krebsforschuini Klin. Onkoiop. 91, 157-164 Berenblum, I 11979) Tumor-formation following treezing with carhon dioxide snow Brit J 1.1/> Pathol 10, 179-184 Berenbllm. 1, i I944i Irritation and carcinogenesis Arc/t. Pathol 38, 233-244 Berman, J J.t Tong. C,T tsn Wn.uams, G, M 11978). 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Ensiron Health 2, 849-871 Gross, N J,, Getz, G S., and Rabinowitz, M. i 1969) Apparent turnover of mitochondrial deoxy ribonucleic acid and mitochondrial phospholipids in the tissues of the rat J Biol Chem. 244, 1552- 1562 I ARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals lo Man (1972). Vol. 1, pp. 95-107 Lowry. 0. H., Rosebrough, N. J,, Farr. A. L . and Randall, R. i (1951). Protein determination with the Folin phenol reagent J Biol. Chem. 193, 265-275 M A L TONI. C,, COTTI, G . MORISI, L.. AND ChIECO, P (1977) Carcinogenicity bioassays of vinylidene chloride. Research plan and early results. Med Lav. 68(4), 241-262. Marmur. j (1961). A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Bud 3, 208-218. Marx, J. L (1978). Tumor promoters. Carcinogenesis gets more complicated Science 201, 515-518. McCormick. J. J. (19791 Evidence that DNA Exci sion Repair Processes in Human Fibroblasts Can Eliminate Potenttidls Cvtoloxtc and Mutagenti Lesions Reported at the 10th Annual Meeting, Environmental Mutagen Society, New Orleans. La , March 8- 12. McKenna, M. J,, Watanabe, P. G . and Gehring,