macromolecular interactions of inhaled methylene chloride in rats and mice

TOXICOLOGYANDAPPLIEDPHARMACOLOGY 93, l-10(1988)

Macromolecular Interactions of Inhaled Methylene Chloride in Rats and Mice

TREVOR GREEN, WILLIAM M. PROVAN, DAVID C. COLLINGE, AND ANNE E. GUEST

Imperial Chemical Industries PLC, Central Toxicology Laboratory, Alderley Park,

Nr Macclesfield, Cheshire SK10 4TJ, United Kingdom

Received June IO, 1987; accepted November 5, 1987

Macromolecular Interactions of Inhaled Methylene Chloride in Rats and Mice. GREEN, T.,

PROVAN,W.M.,COLLINGE,D.C.,ANDGUEST,A.E. (1988). Toxicol.Appl. Pharmacol.93, l- 10. The in vivo interaction of methylene chloride and its metabolites with F344 rat and B&F, mouse lung and liver DNA was measured after inhalation exposure to 4000 ppm [‘%]methylene chloride for 3 hr. DNA was isolated from the tissues 6, 12, and 24 hr after the start of exposure and analyzed for total radioactivity and the distribution of radioactivity within enzymatically hydrolyzed DNA samples. Covalent binding to hepatic protein was also measured. A further group of rats and mice were dosed intravenously with [ “C]formate after exposure to nonradiolabeled methylene chloride for 3 hr to determine the pattern of labeling resulting from incorporation of formate into DNA via the C- 1 pool. Low levels of radioactivity were found in DNA from lungs and livers of both rats and mice exposed to [‘4C]methylene chloride. Two- to fourfold higher levels were found in mouse DNA and protein than in rat. Chromatographic analysis of the DNA nucleosides showed the radioactivity to be associated with the normal constituents of DNA. No peaks of radioactivity were found that did not coincide with peaks of radioactivity present in hydrolyzed DNA from formate-treated rats and mice. Under the conditions of this study there was no evidence for alkylation of DNA by methylene chloride in either rats or Ike. 0 1988 Academic Press, Inc.

Methylene chloride (dichloromethane) is a volatile liquid used as an ingredient in paint stripping and aerosol preparations and as a solvent in a wide variety of industrial applica- tions. A recent National Toxicology Pro- gramme (NTP, 1986) lifetime inhalation bioassay of methylene chloride has shown an increased incidence of lung and liver tumors in B3C3FI mice after exposure at 2000 and 4000 ppm. In the same study there were no corresponding increases in lung and liver tumors in the F344 rat. Previous studies have also failed to elicit this response in either rats or hamsters at similar dose levels by inhalation (Burek et al., 1984) or in mice exposed at lower dose levels in drinking water (Serota et al., 1986).

Two metabolic pathways are known for

methylene chloride, one involving cyto- chrome P-450 mediated oxidation to carbon monoxide, the other glutathione conjugation to give formaldehyde, formic acid, and carbon dioxide (Kubic and Anders, 1975; Ahmed and Anders, 1978). Both have been proposed to involve reactive intermediates potentially capable of interacting with cellu- lar DNA. This potential appears to be real- ized in some short-term mutagenicity tests which utilize microorganisms as their end- point (Jongen et al., 198 1; Green, 1983; Ost- erman-Golkar et al., 1983) but is rarely seen in mammalian cells or in vivo tests (Gocke et al., 198 1; Perocco and Prodi, 198 1; Trueman and Ashby, 1987; Sheldon et al., 1987). Thus although methylene chloride is a lung and liver carcinogen in mice at high-dose levels

1 0041-008X/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

2 GREEN ET AL.

there remains some doubt about the ability of this chemical to cause cancer by a genotoxic mechanism.

As part of a program aimed at investigating the mechanism of carcinogenicity of methylene chloride we have measured the extent of binding of radioactivity to hepatic and pulmonary DNA and to hepatic protein after in- halational exposure of rats and mice to r4C- labeled methylene chloride. Isolated DNA has been hydrolyzed and analyzed by chromatography in order to distinguish between alkylation products and radioactivity incorporated into DNA via the C- 1 pool which results from the metabolism of methylene chloride to formic acid. To facilitate this analysis, rats and mice have been dosed with 14C-labeled formic acid to determine the pattern of radiolabeling of DNA via the C- 1 pool.

METHODS

Exposure. Animals were exposed in the closed recirculating system shown in Fig. I. The glass exposure chamber had an internal volume of 30 liters. Oxygen. temperature, humidity, and carbon monoxide levels were monitored continuously. Carbon dioxide was removed by a trap of carbosorb: carbon monoxide was oxidized with Hopcalite catalyst and trapped as carbon dioxide. Excess water vapor was removed by a cold trap maintained at -20°C. Oxygen levels were maintained automatically from a reservoir and pump linked to the oxygen monitor in the exposure chamber. A 4000-ppm atmosphere of methylene chloride was generated by injection of methylene chloride by syringe into a 250-ml flask maintained at 30°C in the recirculating system. The recirculating atmosphere was maintained at a flow rate of 2 liters/min. The dose was calculated from the weight of methylene chloride required to maintain a 4000-ppm atmosphere for either 40 mice or 4 rats less that required to maintain the same atmosphere for halfthat number of rats or mice. The difference was taken to be the dose received by either 20 mice or 2 rats and was converted to the dose received per kilogram body weight of each species. The use of this procedure eliminated errors due to loss of methylene chloride from the exposure system.

The concentration of methylene chloride in the exposure chamber was analyzed by gas chromatography at 5- to IO-min intervals during the exposure using a 18-m X 4-mm glass column packed with Porapak PS. At 185°C methylene chloride had a retention time of I .5 min.

Materials. Methylene chloride (dichloromethane) ARISTAR grade minimum purity 99.9% was supplied by BDH chemicals (Poole, Dorset. UK). Deoxyribonu- cleic acid (calf thymus, Type I), deoxyribonuclease I (Type III). phosphodiesterase I (Crotalus atrox venom, Type VII), alkaline phosphatase (Type III-R). and prote- ase K (Type XI) were obtained from the Sigma Chemical Co. (Poole, Dorset, UK). Phenol (Analar grade) and m- cresol were supplied by BDH Chemicals Ltd. Phenol so- lutions were each prepared from a fresh unopened batch; m-cresol was vacuum distilled before use. All other chemicals were of the highest purity commercially avail- able.

[“C]Methylene chloride, with specific activities of 19.5.27.5, or 28.8 mCi/mmol was obtained from Impe- rial Chemical Industries PLC, Physics and Radioisotope Services (Billingham, Cleveland, UK). The material had a radiochemical purity of >98% as determined by radio- gas chromatography. [‘4C]Sodium fot-mate, with a specific activity of 57 mCi/mmol and a purity of 98.5% was supplied by Amersham International (Amersham, Bucks, UK).

Animals. Male Fischer 344 rats, 210-255 g body wt. and male B&F, mice, 24-35 g body wt, were housed in temperature-controlled rooms fitted with a 12-hr lighting cycle. Rats were supplied by Charles River Ltd. (Kent, UK) and mice by either Charles River or by Jackson Lab- oratories (Bar Harbor, ME). Feed (PCD Diet, Special Diet Services Ltd, Witham, Essex, UK) and water were provided ad Iibitum except during exposure.

Twelve rats. 2 per group, or 60 mice, 10 per group. were initially exposed to 4000 ppm of [‘4C]methylene chloride, with a specific activity of 83-87 rCi/mmol, for 2 hr. The animals then remained in the closed system for a further 1 hr during which the atmosphere was allowed to decay (to approximately 3000 ppm) as it was taken up by the animals. Following exposure 4 rats and 20 mice were killed at 6. 12. and 24 hr from the start of the exposure period. Lungs and livers were removed and stored at -70°C before extraction of the DNA and protein.

In each experiment an appropriate number of control rats or mice were exposed to air alone.

Thirty-nine mice were also exposed under the same conditions to 4000 ppm [“‘Clmethylene chloride with a specific activity of 450 $Zi/mmol. These animals were killed 6 hr after the start of exposure.

In a further experiment. 8 rats and 40 mice were exposed to 4000 ppm of nonradioactive methylene chloride in a manner similar to that described for [‘4C]- methylene chloride. At the end of exposure, each animal was given a single intravenous injection of [‘4C]sodium formate (1 1 I &i/kg) in deionized water (2 ml/kg) via the tail vein. All of the animals were killed and livers and lungs were removed 12 hr after the start of the exposure period.

DNA and protein e.utruction. DNA from tissues taken from animals exposed to 4000 ppm methylene chloride at the lower specific activity were combined as follows.

DNA BINDING OF METHYLENE CHLORIDE

absorber

FIG. 1. Schematic diagram of the apparatus used to expose rats and mice to [Wlmethylene chloride.

At each time point DNA was isolated from individual rat livers, rat lungs were combined in groups of4, mice livers into groups of 5 or 10, and mice lungs into groups of 20. For the high specific activity experiment livers were combined in groups of 10 and lungs into a single group. Control rat lungs were also pooled into a single group. From 0.4 to 1.3 g of each liver or each group of combined livers were taken for protein extraction (Jollow et al., 1973). DNA was isolated (Irving and Veazey, 1968) from the remaining liver and from the lungs. An additional purification step was added at the end of the published method. The isolated DNA was redissolved in 10 ml of citrate-saline buffer and treated with 200 rg proteinase K for 30 min at 37°C. The DNA was recovered by the addition of 3 M sodium acetate and isopropanol as described by Irving and Veazey (1968). The isolated DNA was washed with 75% ethanol/ 1% sodium chloride solu- tion, 75% (v/v) ethanol, and then with ethanol. Purified samples were assayed for total DNA (Burton, 1956) and contaminating protein (Lowry et al., 195 1).

Measurement of radioactivity. DNA (3-6 mg) or protein (9-16 mg) was weighed accurately, dissolved in 2 ml Soluene, and transferred to a precounted vial containing 10 ml Dimilume scintillator (Packard Ltd). Radioactiv- ity was determined using a Tricarb Model 460 CD liquid scintillation spectrometer (Packard Ltd). Samples were allowed to stand for 24 hr in the dark before counting for 1 hr. Low background glass vials were used throughout the experiments.

Chromafug&zy. After assaying for radioactivity, DNA from either lung or liver and corresponding to the same species, dose, and time point was combined to give samples of approximately 10 mg. To each of these samples was added 75 units ofdeoxyribonuclease and 2 pmol of magnesium chloride in a total volume of 1.5 ml of deionized water. The sample was then incubated at 37°C

for 2 hr before adding 0.25 unit of phosphodiesterase, 8 units of alkaline phosphatase, and a further 2 rmol magnesium chloride. The incubation then continued for a further 18 hr. Following hydrolysis each sample was analyzed by high-performance liquid chromatography using a 20 X l-cm column containing Aminex A6 ion-exchange resin (Bio-Rad Laboratories) and maintained at 55°C. After injection of the sample the column was eluted for 2 hr with 0.32 M ammonium formate buffer (pH 4.35) followed by up to 1.5 hr with 0.4 M ammonium formate buffer (pH 7.0). The flow rate was 1 ml/min. The column effluent was monitored at 254 nm and 3-ml fractions were collected throughout. Under these conditions, typical retention times were 2deoxythymidine, 13 min; 2-deoxyguanosine, 29 min; 2-deoxyadenosine, 57 min; and 2deoxycytosine, 81 min. Trace amounts of the DNA bases were seen in some of the hydrolysates. Their retention times were thymine, 18 min; guanine, 50 min; adenine, 135 min; and cytosine, 142 min. Each hydrolyzed IO-mg DNA sample was diluted with 0.2 M formic acid (1 ml) and analyzed consecutively in three aliquots. Equivalent fractions from the three runs were combined, freeze dried, and 1 ml of distilled water added to each. Ten milliliters of Hionic Fluor scintillator (Packard) was added and radioactivity assayed using the liquid scintillation spectrometer described above. Each sample was counted for 1 hr.

RESULTS

During the initial 2 hr of the exposures to [ 14C]methylene chloride the mean concentrations were 4046 I~I 2 12 ppm for rats and 3897 + 403 ppm for mice. The atmospheric con-

4 GREEN ET AL

TABLE I

RADIOACTIVITY AWXYATED WITH DNA ISOLATED FROM RATS AND MICE EXPOSED TO 4000 ppm [“‘CIMETHYLENE CHLORIDE (83-87 PCIIMMOL)”

Time ON Rat (n = 4) Mouse (n = 20)

Liver dpm/mg hepatic DNA 6 10.5 + 2.8 27.5 f 3.1

12 18.4 f 5.5 28.8 f 5.5 24 16.3 + 1.5 38.2 + 15.2

Lung dpm/mg pulmonary DNA 6 13.0 34.4

12 16.6 68.6 24 22.1 56.1

Note. Results are means f SD for hepatic DNA. Lung samples were combined before analysis. Controls: DNA isolated from both livers and lungs of control animals (n = 22) was found to contain ~1 dpm/mg DNA.

a The dose was calculated to be 5 10 mg/kg for mice and 430 mg/kg for rats.

centrations of methylene chloride in the exposure chamber at the end of the third hour were 2920 + 350 ppm for mice and 3090 i- 156 ppm for rats. These exposures resulted in a dose of 5 10 mg/kg to mice and 430 mg/ kg to rats based on the difference in uptake of dichloromethane from the exposure system when it contained either 40 mice or 4 rats compared to that when it contained 20 mice or 2 rats. The dose of radioactivity to rats was 444 &i/kg and to mice exposed to the higher specific activity dichloromethane, 2700 &i/kg.

Radioactivity was detected in the DNA samples from both rats and mice exposed to radioactive methylene chloride (Table 1). Mouse hepatic and pulmonary DNA contained two- to fourfold more radioactivity than the equivalent samples from rats. DNA from mouse lungs contained up to twice as much radioactivity as that from mouse liver. In the rat, the levels were similar in both tissues (Table 1). For mice dosed with [14C]- methylene chloride at the higher specific activity (450 pCi/mmol) and killed 6 hr after the start of the 3-hr exposure an eightfold in-

crease in the radioactivity bound to DNA was seen compared to DNA from mice dosed at the lower specific activity. Liver DNA from mice exposed to the higher specific activity material contained 236.8 dpm/mg and lung DNA 279.7 dpm/mg.

DNA isolated from the lungs and livers of both test and control rats and mice contained 0.5-l .5% of protein as measured by the method of Lowry et al. ( 195 1). The background level of radioactivity associated with DNA from control rats and mice was 4.86 f 2.5 dpm for a mean weight of DNA assayed of 5.22 + 0.14 mg (n = 22), i.e., less than 1 dpm/mg DNA.

Radioactivity was also associated with hepatic protein after exposure to 4000 ppm [‘4C]methylene chloride. As with DNA. mouse hepatic proteins contained 2-3 times more 14C than rat hepatic proteins (Table 2). Peak binding levels were seen at 6 hr in the mice and 12 hr in the rat.

Radioactivity was incorporated into DNA following a single intravenous dose of [‘“Cl- formate (Table 3). DNA from rat tissues contained higher levels of radioactivity than the equivalent mouse DNA. In both species 2-3 times more radioactivity was found in DNA isolated from the lung than in DNA from the liver. The level of radioactivity associated with mouse liver protein in this experiment was 401.5 ? 88.6 dpm/mg protein (n = 4).

Chromatography of hydrolyzed DNA from [14C]formate-dosed rats and mice showed peaks of radioactivity eluting with 2- deoxythymidine, deoxyguanosine, and deoxyadenosine as a result of incorporation of radioactivity into these nucleosides from the C-l pool (Fig. 2). Hydrolyzed DNA from both livers and lungs of rats and mice exposed to [‘4C]methylene chloride gave a qualita- tively similar distribution of radioactivity, all of the peaks being coincident with those seen in formate treated animals. The chromato- grams of hydrolyzed lung and liver DNA from mice exposed to [‘4C]methylene chloride at the highest specific activity are shown in Figs. 3 and 4, respectively.

DNA BINDING OF METHYLENE CHLORIDE 5

TABLE 2

RADIOACTIVITY ASSOCIATED WITH HEPATIC PROTEIN FROM RATS AND MICE EXPOSED

TO 4000 ppm [‘4C]M~~~~~~~~ CHLORIDE (83-87 &I/MMOL)’

Time 0-4

Rat (n = 4) Mouse (n = 4)

nmol/mg nmol/mg dpm/mg protein dpm/mg protein

6 306.9 + 24.1 1.59+0.12 1054.9 f 129.8 5.66 k 0.62 12 376.7 + 43.7 1.95 + 0.23 927.6 f 116.0 4.90 * 0.50 24 231.5 + 18.8 1.20~0.10 655.1 + 96.7 3.46 + 0.44

Note. Results are expressed as means + SD. ’ The dose was calculated to bc 5 10 mg/kg for mice and 430 mg/kg for rats.

The peak of radioactivity not retained by the column and eluting in the void volume in fraction 2 in both methylene chloride- and formate-treated animals is believed to be due to protein contamination of DNA. Because of the higher levels of radioactivity bound to protein (Table 3), contamination of the DNA by protein at the 1% level would be consistent with the levels of radioactivity eluted in the void volume.

DISCUSSION

The binding of radioactivity to highly purified DNA after exposure to radiolabeled test

TABLE 3

INCORPORATION OF RADIOACTIVITY INTO HEPATIC AND PULMONARY DNA OF RATS AND MICE FOLLOW-

INGA SINGLE I.V. DOSE OF [“%]FORMATE

dpm/mg DNA

Organ Rat (n = 8) Mouse (n = 40)

Liver a 40.15 + 7.75 28.32 k 8.40 Lungb 132.11 78.06

Note. Rats and mice were exposed to nonradioactive methylene chloride (4000 ppm) for 3 hr prior to dosing with [‘4C]formate (111 pCi/kg). All of the animals were killed 12 hr after the start of the exposure.

’ Liver results are means + SD. b Lung results are from pooled samples.

compound is frequently used as a measure of its potential to induce cancer by somatic mu- tation. Peak binding of radiolabel usually oc- curs within 24 hr of exposure to a single dose of chemical (Lutz, 1979). For this reason binding to DNA was measured 6, 12, and 24 hr after the start of exposure to methylene chloride.

The DNA isolated from rats and mice exposed to [ 14C]methylene chloride was, as ex- pected, found to contain radioactivity at all time points. Two possible mechanisms have been proposed to account for this radioactivity, one involving alkylation by a reactive me- tabolite, possibly either the glutathione con- jugate of methylene chloride or formyl chloride, the other involving incorporation of [‘4C]formate produced by methylene chloride metabolism into the normal pathways of DNA biosynthesis (Fig. 5). Careful analysis of the DNA from [‘4C]methylene chloride-exposed rats and mice has revealed that the radioactivity associated with DNA results from incorporation of formate via the C- 1 pool. No evidence was found for the existence of abnormal nucleosides which might suggest alkylation by reactive methylene chloride metabolites.

The two- to fourfold higher levels of radioactivity found in mouse pulmonary and hepatic DNA possibly reflect either increased metabolism of methylene chloride in the mouse or a higher rate of DNA synthesis, or

GREEN ET AL.

60

J 20 30 40 50

Fraction Number

FIG. 2. Chromatographic analysis of hydrolyzed DNA from livers of mice exposed to nonradiolabeled methylene chloride and given a single i.v. dose of [‘4C]formate (100 &i/kg). The upper trace is the uv absorbance at 254 nm; the lower trace shows radioactivity. Peaks marked dT, dG, dA, dC, G, A, and C correspond to the retention times of 2-deoxythymidine, 2deoxyguanosine, 2-deoxyadenosine, 2deoxycy- tosine, guanine, adenine, and cytidine, respectively.

a combination of both in the smaller, more reflect a higher rate of metabolism of methy- metabolically active species. The lower incor- lene chloride in the mouse. The high specific poration of [14C]formate into mouse lung activity [‘4C]formate was used to radiolabel and liver than into the rat tissues may again an existing body burden of unlabeled formate

1,000

900

600

F 700

s $ 600 ‘C 0 ; 500 5 .c .g

400

.i 300

z 200

Fraction Number

FIG. 3. Chromatographic analysis of hydrolyzed DNA from the lungs of mice exposed to 4000 ppm [‘4C]methylene chloride (450 $Zi/mmol). The upper trace is the uv absorbance at 254 nm; the lower trace shows radioactivity. Peaks marked dT, dG. dA, dC, G, A, and C correspond to the retention times of 2- deoxythymidine, 2-deoxyguanosine, 2-deoxyadenosine, 2deoxycytosine, guanine, adenine, and @dine, respectively.

DNA BINDING OF METHYLENE CHLORIDE

n

CIA

dG dC

B

1 10 20 30 40 50 60 70

Fraction Number

FIG. 4. Chromatographic analysis of hydrolyzed DNA from livers of mice exposed to 4000 ppm [“Cl- methylene chloride (450 pCi/mmol). The upper trace is the uv absorbance at 254 nm; the lower trace shows radioactivity. Peaks marked dT, dG, dA, dC, G, A, and C correspond to the retention times of 2- deoxythymidine. 2-deoxyguanosine, 2deoxyadenosine, 2-deoxycytosine, guanine, adenine, and cytidine, respectively. Fractions 2 and 3 attenuated IO-fold.

produced by exposure to unlabeled methylene chloride. Higher concentrations of nonradioactive formate present in the mouse as a result of greater methylene chloride metabolism could result in an overall lower specific radioactivity than in the rat and thus in incorporation of lower levels of radioactivity into DNA. It must be emphasized that the formate experiments were designed only to es- tablish the labeling pattern of DNA via the C-l pool and were not intended to, nor is it

possible, to make direct quantitative compar- isons between species or with [ 14C]methylene chloride-exposed animals.

In addition to identifying C-l incorporation the chromatographic techniques allow detection of abnormal constituents in DNA. Previous studies have established that the type of chromatography used in this study is able to detect a variety of DNA adducts which typically elute with, or more usually later than, the normal constituents of DNA

Cl$Cl, ,sm GSCH,CI- GS@=Cl& LGSCH@H -GSH + HCHO -DNA I I 4 I

DNA HEOOH -C-l -DNA

I t

co2

02 CH,CI, =HCOCl -CO l WI

I DNA

FIG. 5. The metabolism of methyiene chloride and the possible interactions of its metabolites with DNA.

8 GREEN ET AL.

(Singhal, 1974; Green and Hathway. 1978; Laib and Bolt, 1978). In this study each analysis was continued for 2 hr after the elution of the normal nucleosides and for the last 1.5 hr at a buffer concentration and pH chosen to elute all possible adducts of DNA nucleosides and bases from the column. This was con- firmed by the high recovery of radioactivity (range 97- 104%) from the chromatography of mouse lung and liver deoxynucleosides.

Only low levels of radioactivity were found in DNA from animals exposed to 4000 ppm methylene chloride at a specific activity of 83-87 &i/mmol. In order to further increase the sensitivity of the chromatographic analysis to detect any alkylation within these low levels, which include C-l incorporation, a further study was undertaken in mice using a fivefold increase in specific activity of the methylene chloride. The ability of a DNA binding study to detect alkylation of DNA by a chemical is dependent on the amount of radioactivity administered to the animal and on the amount of DNA isolated for the assay of bound radioactivity. The relationship between dose and the amount of the chemical bound to DNA may then be expressed as the covalent binding index (CBI) (Lutz, 1979). Based on the dose to mice (total body weight, 1200 g) of 2,700 &i/kg body wt and the recovery of approximately 50 mg of hepatic DNA and 10 mg of pulmonary DNA the minimal CBI was 0.002 for the liver and 0.0 1 for the lung. Because of the necessity to ana- lyze the DNA by chromatography this level of sensitivity assumes that any alkylation product of DNA would be collected in a single fraction. The sensitivity of the study would have been sufficient to detect alkylation of DNA at levels below those associated with even weak carcinogens (Lutz, 1979). Po- tent carcinogens such as dimethylnitros- amine and alkatoxin Bl are reported to give CBI values in excess of 10,000.

The appearance of radioactivity in the void volume of the column has been reported in similar studies with a wide variety of chemicals (Bergman, 1982, 1983; Stott et al., 1982;

Laib and Bolt, 1978). This activity has been attributed to protein contamination or to un- hydrolyzed oligonucleotides or it may result from the breakdown of unstable DNA adducts. Repeated hydrolysis of the same sample for different incubation times did not alter the level of radioactivity, suggesting that it was not due to oligonucleotides. Radioactiv- ity was also present in the void volume of the DNA hydrolysates from both mice (Fig. 2) and rats dosed with [‘4C]formate suggesting that this radioactivity is not due to the breakdown of DNA adducts nor is it toxicologi- cally significant.

The hepatic protein binding figures (Table 2) for [14C]methylene chloride-dosed rats and mice were high relative to both DNA (50X) and to protein from [‘4C]formate-dosed mice. This suggests that methylene chloride or its metabolites bind covalently to proteins in addition to being incorporated via the C- 1 pool. This would be consistent with the results from in vitro studies with methylene chloride reported by Anders et al. ( 1977). The high specific activity of the protein associated with the DNA samples in the present study could account for the levels of radioactivity seen in the void volumes. Furthermore the observation of this radioactivity in many hepatic DNA binding studies of a wide variety of unrelated chemicals suggests a common origin such as contamination by radiolabeled proteins. The quantity of radioactivity eluted in the void volume during the analysis of hepatic DNA samples was greater than that seen during the analysis of lung DNA. A similar difference between hydrolysates of hepatic DNA and DNA isolated from other tissues has been reported previously and is believed to reflect the cell specificity shown by chemicals in extrahepatic organs (Bergman, 1983). In this respect methylene chloride appears to be specific for a single cell type in the mouse lung, the nonciliated or Clara cells (Green et al., 1986).

In conclusion, under the conditions of this study there was no evidence for alkylation of DNA by reactive metabolites of methylene

DNA BINDING OF METHYLENE CHLORIDE 9

chloride suggesting that a single exposure to methylene chloride is not genotoxic in either the lungs or liver of F344 rats or B&F, mice. This conclusion is in agreement with other tests which have failed to detect any evidence of genotoxicity in animals in vivo (Gocke et al., 198 1; Sheldon et al., 1987; Trueman and Ashby, 1987). Most in vitro mammalian cell mutagenicity assays of methylene chloride are also negative (Burek et al., 1984; Jongen et al., 198 1; Perocco and Prodi, 198 1) and even in those tests (such as chromosomal ab- errations) (Thilager and Kumaroo, 1983) this activity is not reproduced in vivo (Gocke et al., 1981; Sheldon et al., 1987; Burek et al., 1984). Methylene chloride appears to be con- sistently mutagenic only in Salmonella, where evidence has been obtained to suggest that this is due to the ability of the bacteria to metabolize methylene chloride and to differ- ences in the structure of bacterial cells to those of mammalian cells (Green, 1983). Consequently it was proposed that the results in bacteria were not relevant to the in vivo exposure of animals or man to methylene chloride, a conclusion supported by the present work. Although it cannot be ruled out that repeated exposure to dichloromethane could result in DNA alkylation, it is surprising that this study and the whole range of mammalian short-term tests which have been conducted using a variety of single dose levels and routes of administration have all failed to find evidence of genotoxicity. There appears there- fore to be a significant body of evidence, in- cluding this study which used the species and dose levels used in the NTP bioassay, to suggest that methylene chloride is not genotoxic in rats and mice in vivo.

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macromolecular interactions of inhaled methylene chloride in rats and mice

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