Exp. Path. 26, ilil 111.-, il~IK~)

MRC Pneumo('oniosis Unit, Llandough Hospital, Ppnarth, Glamorgan, United Kingdom

The metabolism of benzo(a)pyrene by the lungs of asbestos exposed rats

By R. C. BROWN, A. POOLE and U. T. A. FLE3UNG

With one figurr

(Received February G, 1984)

Address for correspondence: Dr. R. C. BROWN, i\lRC Pneumoconiosis Unit, Llandough Hospital, Penarth, Glamorgan CF6 1XW, United Kingdom

Ke y words: benzo(a)pyrene; lung; asbestos, pathogenicity; crocidolite

Summary

A group of Fischer F344 rats were exposed to an aerosol of crocidolite asbestos for 36 d. Short-term organ cultures were established from the lungs of these animals and from a similar control group. The ability of these cultures to metabolise benzo(a)pyrene to water­soluble and ether-soluble forms was measured. Crocidolite treatment reduced the lungs ability to produce both types of metabolite although only the reduction in water-soluble forms was significant. DNA binding increased in cultures from treated rats, though this was not statistically significant. The possible relationship of these results to asbestos pathogenesis is discussed.

Introduction

Asbestos exposure is a cause of lung cancer: it also interacts with cigarette smoking to increase the incidence of this tumour in smokers exposed to this mineral. The whole subject of cancer risks and asbestos exposure has been the subject of many reviews but the position is described particularly clearly in ACHESON and GARDNER (1979, 1983). In experimental animals asbestos also causes mesothelial and lung tumours (see for example WAGNER et al. 1974) but the interaction with cigarette smoking has not been demonstrated in such studies. However a more than additive interaction between asbestos fibres and chemical carcinogens has been demonstrated in several short term experiments (see for example BROWN et aI. 1983; REISS et al. 1983 and review by MOSSMAN et aI. 1983).

A number of hypotheses have been put forward to explain these phenomena (see Moss­MAN et al. 1983) the two main possibilities being that the fibres either enhance the uptake or retention of a carcinogen or alter its metabolism. Studies with cultured cells in this laboratory (POOLE et at 1983) have shown a reduction in the formation of water-soluble metabolites from benzo(a)pyrene (BP) and the reduced formation of the glucuronide from I-naphthol (BROWN et aI. 1983b).

To test the hypothesis that asbestos exposure could reduce xenobiotic detoxification in vivo as well as in vitro a group of rats were exposed to crocidolite and the metabolism of BP was studied in short term organ cultures derived from the lungs of these animals.

111 atel'ials and }[ ethods

Ani mal p x p os UIP: Twenty male, barrier maintained, Fischer F344 rats were randomly aIIo­cated to 2 groups. The test group was caged in an inhalation chamber (TIMBRELL et al. 1970) and exposed to a dust eloud containing an aerosol of the UICC standard of crocidolite asbestos (TIMBRELL and RENDALL 1971). Dusting took place for 26 d for approximately 61/2 h a day giving a final cumula­tive dose of 4.512 g/m~. After dusting the rats remained in the chamber for one week and were then returned to normal living quarters. Th!' eontrol group r{'mained in normal quarters throughout.

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Post-PXf!OSlllt' trt'atJllt'llt: f'rt'liminary l'xpl'riments revl'aleu that in order to dptl,et any hydrocarbon nH'tabolism in lun?:s from this strain of rats some step had to be taken to induce mixed function oxidase aetivity. :Vleasurabll' and consistent activity towards benzo(a)pyrene was obtained by adding phenobarbitone-sodium (0.05 °0) to the drinking water for one week. This induction rrgime was therefore uSl'd in all subsequent studies.

After one week on phenobarbitone the animals were killed by intraperitoneal pentabarbitone overdose. The lungs were excised, sl'parated from the major airways and weighed. As this strain of rats has a very high ineidenep of leukaemia the liver and spleen were taken for histology, one control animal showed signs of this diseasl' and has not been included in the results.

Preparation of organ cultures: The excised lungs were washed thoroughly in Dulbecco's modified Eagles medium (DME:\f) to remove blood. The left and right lungs were separated and cut into 2 mm cubes whieh werr evrnly divided between 2 sterile bottles for each lung: that is 4 cultures for each animal. The lung pirees were washed again with DMEM and 15 ml of DMEM containing 15 % heat inactivated foetal bovine serum and a mixture of tritiated and "cold" benzo(a)pyrene was added to each bottle. The DP was purified before use by eluting from a SepPak silica gel cartridge (WATERS Associates) with petroleum ether and added as an acetone solution to a final concentration of 10,uCi (3 ,ugl/ml; the final acetone concentration being 0.5 %. The bottles were gassed with 8% carbon dioxide in air to adjust thl' pH of the medium to 7.2 and then incubated at 37°C in a shaking water bath.

Estimation of DP metabolism: At 2, 6 and 24 h after the start of incubation 1 ml samples of medium were taken from each bottle and placed in 2 ml of ice-cold ether in stoppered tubes; these were gassed with nitrogen and stored at --70°C until all the samples were collected. At 24 h the remainder of the medium was also treated with nitrogen and frozen. The tissue was then washed in ice-cold phosphate buffered saline and homogenised in Tris-EDTA buffer (pH 8) using an Ystral XI020 homogenizer. The homogenates were also gassed and frozen at -70°C.

The medium samples were extracted 3 timl's with twice their volume of water-saturated diethyl ether: the water soluble metabolites were then estimated by counting 200,u1 of the aqueous phase in 4 ml of Instagel (Packard Instrument Co) in an Intertechnique SL4000 scintillation counter with on-line queneh correction using the' external standard method. The ether extracts were pooled and BP separated from its metabolites using thin layer chromatography by the method of MACNICOLL et al. (1980).

The tissue homogenate was well mixed and a 1 ml sample taken, diluted and its protein content measured using the method of LOWRY et al. (1951). The DNA was isolated from half the remaining homogenate using the method of TIERNEY et al. (1977). An aliquot of the isolated DNA was counted as described above and the amount of D~A estimated using the diphenylamine method. The reduced glutathione content of the homo?:enate was then estimated using the method of SWADITAT and TSEN (1972).

Statistical significant'l' was assessed by the use of Student's t-test treating each culture as a sepe­rate sample.

Results

Lung wet weight and protein content increased after asbestos exposure. While the increase in wet weight was not statistically significant (whether expressed in absolute terms or as a proportion of the body weight) the increase in protein content was significant at the 5 % level (table 1).

Table 1

Treatment

Asbestos Control

Wet lung wt (g)

2.06 ± 0.37 1.94 ± 0.26

Lung as % body wt

0.48 ± 0.05 0.44 ± 0.03

Effeet of asbestos exposure on lung size and composition. * significance at 5 % lev .. l

Protein mg/lung

97.3 ± 7.0 79.6 ± 10.8*

Table 2 :Jletabolism of eH)-benzo(a)pyrenp by cultured lung from asbestos exposed and control rats

Treatment H20 soluble Ether soluble Binding of (3H) metabolites metabolites -DP to DNA nmol/g Wl't wt. nmol/g wet wt. pmol/.ug DNA

Control 120.9 ± 37.9 15.7 ± 7.0 79.5± 52.8 Asbestos 80.fJ ± 14.9 10.5 ± 1.21 106.0 ± 82.5

Lung tissue from asbestos exposed and ('ontrol rats were incubated with (3H) benzo(a)pyrene for 24 h. Metabolites were separated and e'xtractl'd and DXA binding estimated as described in the text.

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Fig. 1. The production of water soluble metabo­lites from benzo(a)pyrene measured as described in the text. The means and standard errors are shown from the control animals (solid line) and crocidolite exposed (dotted line).

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24

The lungs of both asbestos exposed and control rats metabolised tritiated BP to organic and water soluble metabclites; with Ecsbestos treatment apparently reducing the ability of the lung to metabolise this compound (table 2). Only the difference in the production of water soluble metabolites was significrcnt, cultures from dusted animals produced less of this type of metabolite and this was significant whether expressed on the basis of protein (fig. 1) or wet weight (table 2).

The DNA isolated from all cultures was radioactive even after extensive washing and this was presumably due to covalent binding of BP metabolites. The level of this binding was increC1sed in the asbestos treated lungs and was the only measure of metabolism to change in this direction. However the variability in labelling prevented the difference reaching significance (table 2).

The level of reduced glutathione was lower (116 ± 4.2 nMol GSHjmg protein) in the asbestos exposed group than in the controls (85.2 ± 3.6) and this difference is significant at the 5 level.

A preliminary study was undertaken in which the ether soluble metabolites were analysed by HPLC. This revealed no differences in the pattern of metabolites produced in the two sets of cultures and the data is not shown in this present paper.

Discussion

The lungs of oosbestos exposed rats appeared less able to metabolise BP than those from control rats, with a reduction in both organic and water soluble metabolites. However only the difference in water soluble metabolites reached statistical significance (p < 0.05). This would appear to confirm our hypothesis that phase two (conjugation) reactions are inhibited by asbestos exposurr. However the (albeit non-significant) reduction in ether soluble meta­bolites suggests that there may have been less overall metabolism, though if this were the case the increC1se in DNA binding would not be explained.

It has been suggested that asbestos pathogenicity is mediated by the production of reac­tive oxygen radicals (see for example DOLL et al. 1982) and that these could also be responsible for the oxidation of some xenobiotics (ROMAN-FRANCO 1982). The decrease in the level of reduced glutathione which we' found in the treated animals might be due to the detoxification of these radicals or their produets. If such a phenomenon reduced the pool of GSH sufficiently this ('oulel result in thp redu('rd conjugation of some hydrocarbon metabolites and this would

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be particularly tnlP ot ""('I! I) ~.\ binding' "lwcip,; as "lIlIlP l'poxides. Howenr we fl'el that the rt'duction ill watl'r solllhll' Illetabolitps is IllllikPl~' to be dut' to a loss of glutathiollP ('olljug'ates alolle, especially as wp have reportpd thp reduerd formation of glucuronidrs in ('ultures of various cell t~'pes after asbpstos pxposure.

Pulmonary drug metabolism has been reported to be insensitive to phenobarbitone induc­tion (GRAM 1980); this may br due to most la,boratory animals already having fully induced pulmonary mixed function oxid(lsr activity (BENFORD and BRIDGES, in press). The strain of rats used in this experiment kept undrr our laboratory conditions have no detectable pul­monary BP oxidising a<"tivity unless some step is taken to induce this activity. 'While we have found phenobarbitonp to be thr most reliable inducer; it is possible that the dust exposed animals have an altered response to this compound, with the balance between oxidation and conjugation being perturbed. The results of this preliminary experiment suggest that, if the mechanism (if asbestos c(lrcinogrnesis is to hr understood, it would be fruitful to earry out further work on the uptake and metabolism of hydrocarbons in dusted animals.

There ne sevpral p()~sible explanations for our findings but when taken in combination with our results with ('pll lint's (BROWN et a1. 1983 b; POOLE et a1. 1983) the results in this present paper lend support to the theory that asbestos exposure reduces phase two meta­bolism. In future studirs the nature of the water soluble metabolites must be examined espe­cially in view of the reports of speciPR differences in the formation of glueuronides in lung tissue (MOORE and COHEN 1978; COH EN ot a1. 1981).

Acknowledgements

Dr. J. C. WAGNEH, Mr. J. W. SKIDMOHE and Mr. R. J. HILL are thanked for providing the dusting' facilities. The authors also wish to thank Dr. P. H. EVANS and Mr. C. J. TUHVER for their technical assistance, Mr. Cu Y for producing th~ illustration and Mrs. J. LEEKE for typing the manuscript.

Literature

ACHESON, 1£. D., and M. J. GAHDNEH, The ill effects of asbestos on health. Asbestos, vol. :2. Final report of the advisory committee. HMSO, London 1979.

- - Asbestos: The control limit for asbestos. HMSO, London 1983. BENFOHD, D. J., and J. W. BRIDGES, Xenobiotic metabolism in lung. In: Progress in Pulmonary

Toxicology (HOOK, G. 1£. R., BROWN, R. C., and YOKOYAMA, E., editors). Elsevier/North-Holland vo!' 1, in press.

BROWN, R. C., A. POOLE and G. T. A. FLEMING, The influence of asbestos dust on the oncogenic transformation of C3HlOTI/2 cells. Cancer Letters 68, 221-227 (1983).

- G. T. A. FLEMING and A. KNIGHT, Asbestos affects the in vitro uptake and detoxification of aro-· matic compounds. Environm. Health Perspect. 1)1, 315-318 (1983 b).

COllEN, G. M., 1£. M. GIBBY and R. MEHTA, Routes of conjugation in normal and canc{']"ous human lung. Nature 291, 662-664 (1981).

DOLL, N. J., R. P. STANKl'S, S. GOLDBACH and J. 1£. ALVAGGIO, In vUro effed of asbestos fibres on polymorphonuclear leUkocyte function. Int. Arch. Allergy appl. Immun. 68, 17--21 (1982).

GRAM, T. E., The metabolism of xenobiotics by mammalian lung. In: Extrahepatic Metabolism of Drugs and Other Foreign Compounds (GRAM, T. E., editor). MTP Press Ltd., Lancast(,I, England 1980, pp. 159-209.

LOWHY, 0. H., N .. J. ROSEBROllGH, A. L. FARR and R. J. RANDALL, Protein measurement with the Folin phenol reagent. BioI. Chern. 193, :265-275 (1951).

l\iACNICOLL, A. D., P. L. GROVER and P. SIMS, The metabolism of a series of polycyclic hydrocarbons by mouse skin maintained in short term organ eulture. Chern. BioI. Interactions 21, 168-188-(1980).

MOORE, B. P., and G. M. COHEN, :\Ietabolism of benzo(a)pyren~ and its major metabolites to ethyl acetate-soluble and watN-solubll' metabolites by cultured rodent trachea. Cancer Res. 38, 3066-3075 (1977).

MOSSMAN, B., W. LIGHT and E. WEI, Asbestos: Mechanisms of toxicity and carcinogenidty in the r~spiratory trad. Ann. Rev. Pharmacol. Toxicol. 23, 595-615 (1983).

POOLE, A., R. C. BHOWN and G. T. A. FLEMIXG, Study of the cell transforming ability of amosite and crocidolite and the ability to induce changes in the metabolism and macromolecular binding of benzo(a)pyr~np in C3HIOTI!Z ('('lis. Ellvironm. Health P(·rspcct. 1)1, 319-324 (1983).

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REISS, B., C. TO'\li, ;-;. T'·:LI.\(i and n. :'11. WILLLD1S, Enhanceml'nt of bPJlzo(a)pyrenc mutagenicity by ehrysotile asbestos in rat liver l'pithplial cells. Enyiron. Res. 31, 100-104 (1983).

ROMAN-FR.\)[co, A. A., ~on-l'nzymatie l'xtramicrosomal bioaetivation of chemical carcinogens by phagocytes: a proposed nl'W pathway. J. Theoret. Biol. 97, 543-555 (1982).

SWADITAT, A., and C. C. TSE)[, Dt·trrmining simple sulfhydryl compounds (low molecular weight) and their eontents in bioiogieal samples by Ilsing 2,2'-dithiobis-(fi-nitropyridine). Analyt. Biochem. 45, 349-:346 (1972).

TIERNEY, B., A. HEWEH, C. W"\LSI!, P. L. Gl((WER and P. SIMS, The metabolic activation of 7-methvl-benz(a)anthraeenc in mousl' skin. Chern. Biol. Interactions 68, 179-193 (1977). "

TIMBRELL, V., .I. W. SKIDMORE. A. W. H YETT and J. C. W.W2'!EH, Exposure chambers for inhalation experiments with standard refercme samples of asbestos of the International Union Against Cancer (UICe). Aerosol Sei. 1, 215-220 (1970).

- and R. E. G. RENDALL, Preparation of the elCC standard reference samples of asbestos. Powder Techno!. 5, 279--287 (1971).

WAGNER, J. C., G. BERI\Y, V. TmRRELL and J. W. SKIDMORE, The effects of the inhalation of asbe­stos in rats. Brit. J. Caneer 29, 2.'i2--2fi9 (1974).

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