Critical genes as early warning signs: example of vinyl chloride

Toxicology Letters 102]103 Ž1998. 603]607

Critical genes as early warning signs: example of vinyl chloride Marie-Jeanne MarionU INSERM, Unite´ 271, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France

Abstract We have analysed liver angiosarcomas from individuals having been occupationally exposed to vinyl chloride ŽVC. to identify, in cancer-related genes, lesions which could be VC-specific. Two genetic alterations have been identified: the first one is a GGC ª GAT ŽGly ª Asp, Asp13p21. mutation at codon 13 in the Ki-ras gene, found in five out of six tumors. The second one is an ATª TA transversion in the p53 gene resulting in missense mutations at different codons and was found in three out of six tumors. By analysing both the tumors and sera from the same patients, we have shown that the Asp13p21 and mutant p53 proteins could be detected reliably in the serum. We thereafter analysed 225 serum samples, selected from a cohort of about 900 VC-exposed workers, for the presence of the two mutant proteins and p53 antibodies. A statistical analysis supports a strong dose-response relationship between the serum markers positivity and the VC-exposure. A follow-up of this cohort should now allow us to assert the predictive value of these markers. Q 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vinyl chloride; Occupational exposure; Angiosarcoma; p21ras ; p53; p53 antibodies

1. Introduction Vinyl chloride ŽVC. is a gas used in the plastics industry to produce polyvinyl chloride ŽPVC.. Exposure of humans to VC occurs by inhalation essentially at the workplace, especially in VC polymerisation plants. VC has begun to be used at an industrial scale to produce PVC in the 1930s. But it was really after 1950 that PVC

U

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production expanded: 1.5 million tons in 1950, 12 million tons in 1985, 21.5 million tons in 1996 worldwide. The first effects of VC on human health were noted in the 1930s. At several thousand ppm, VC reacts with the central nervous system. By 1945]1955, other effects, including scleroderma, fibrosis and enlargement of the liver and spleen, acro-osteolysis and Raynaud syndrome, were then recognised as resulting from chronic exposure at levels of about 1000 ppm Ž‘vinyl chloride disease’.. VC was suspected to be carcinogenic since 1971 by Viola et al. Ž1971. and that was confirmed in

0378-4274r98r$ - see front matter Q 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274Ž98.00255-0

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M.-J. Marion r Toxicology Letters 102]103 (1998) 603]607

1974 by Maltoni and Lefemine Ž1974. who described angiosarcomas of the liver ŽASL. among other tumors, in rats exposed to VC. The same year, the retrospective discovery of three deaths by ASL in the same PVC production plant was reported ŽCreech and Johnson, 1974.. The causal relationship between exposure to VC and ASL has been since then well established by epidemiological studies. Since 1974, 197 cases of ASL have been reported in industrial countries. VC is activated by the microsomal cytochrome P450 mono-oxygenase system into chloroethylene oxide ŽCEO. which rearranges spontaneously into chloroacetaldehyde. VC and CEO induce mutations by base-pair substitutions in various test systems, suggesting that VC acts through the formation of miscoding DNA adducts. Among the four known DNA adducts, 1, N 6-ethenoadenine, 3, N 4-ethenocytosine and N 2 ,3-ethenoguanine are promutagenic in vitro ŽBarbin et al., 1981.. We have undertaken the molecular analysis of human ASL related to VC-exposure in order to identify, in genes known to be involved in cellular transformation, lesions which could be VCspecific. Two genetic alterations, which should meet such a criterion, have been identified, in the Ki-ras oncogene and p53 tumor suppressor gene. Oncoproteins and p53 antibodies have been detected in sera from cancer patients. We have thereafter investigated, using immunological techniques, the presence of Asp13p21, mutant p53 protein and p53 antibodies in blood of VC-exposed workers to test the hypothesis that these mutations are early genetic lesions specific of the carcinogen and to see how useful this finding may be for earlier recognition of individuals at highest risk of developing angiosarcoma. 2. Identification of genetic lesions carcinogenspecific in cancer-related genes 2.1. Ras gene analysis In a first experiment, six tumors were analysed. None of the six tumors exhibited a mutation at codons 12 and 61 in any of the three ras genes, nor at codon 13 for Ha-ras and N-ras. DNA from five tumors, hybridised strongly with a

-CCTACGCCACCAGCTCCAAAC- oligonucleotide probe complementary of the sequence of the Ki-ras proto-oncogene and with a probe in which the normal codon -GCC- Žnon-coding strand. had been replaced by -GTC-. DNA from peritumoral tissue or from lymphocytes from the same patient exhibited the normal sequence, indicating that the observed mutation was somatic ŽMarion et al., 1991.. The mutation was confirmed by sequencing. Additional human VCrelated ASL have now been analyzed; this very same mutation was found in 15 tumors out of 18 ŽMarion, unpublished results .. The specificity and high prevalence of the GGC ª GAC Ki-ras gene mutation we showed here are consistent with the well defined etiology of these angiosarcomas and point to an early genotoxic event. Indeed, in man, mutations at codon 13 seem relatively rare. They occur, for example, only in 10% of lung and colon carcinomas carrying a mutated Ki-ras gene. Also in spontaneous human tumors, the mutations are quite diverse, involving either the first or second base of the codon, and leading to the incorporation of various amino acids. On the contrary, specific mutations associated with high prevalence are common in experimental chemical carcinogenesis. On the other hand, only a few sporadic and Thorotrast-induced ASL contained a point-mutated Kiras gene and the mutations were essentially found at codon 12 ŽPrzygodzki et al., 1997.. This G ? C ª A? T transition is also consistent with the known mutational pattern induced by VC in bacteria and human fibroblasts which shows a majority of G ? C ª A? T transition ŽBarbin et al., 1985; Matsuda et al., 1995.. 2.2. p53 gene analysis Five frozen tumors and one cell line established from a liver angiosarcoma were investigated for the presence of mutations in the p53 gene in exons 5]8 ŽHollstein et al., 1994.. Three Aª T missense mutations were found, one at codon 249 ŽAGGª TGG, Argª Trp., one at codon 255 ŽATCª TTC, Ile ª Phe. and one at codon 179 ŽCATª CTT, His ª Leu.. These p53

M.-J. Marion r Toxicology Letters 102]103 (1998) 603]607

mutations were tumor-specific since they were absent from non-tumoral tissues from the same patients. 3. Detection of oncoproteins in the serum of workers exposed to VC In a preliminary experiment, we have shown by immunohistochemistry, using a monoclonal antibody specific of the Asp13p21 protein, the presence of the mutated protein in all the tumors in which a mutated Ki-ras gene was found. For the same patients, the Asp13p21 protein was also found in the serum. Also the mutant p53 protein was detected in the serum of the patients having developed a tumor containing a mutated p53 gene. In most cases, p53 antibodies were found in the same sera ŽTable 1.. In two pilot-studies, we have shown the high frequency of the Asp13p21 in the serum of a group of workers heavily exposed Ž29 positive sera out of 52. ŽDeVivo et al., 1994; Brandt-Rauf et al., 1995. and that four samples of serum from workers without liver cancer, out of a group of 77 workers, contained anti-p53 ŽTrivers et al., 1995.. Now, from a cohort of 900 VC-exposed workers, 218 subjects without cancer, plus seven liver cancer cases Žsix ASL and one hepatocellular carcinoma., have been selected to be tested for the three markers. The exposure has been esti-

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mated in parts per million-years Žppm-years. using the exposure matrix of Heldaas et al. Ž1984. and the subjects selected on the estimated exposure to get four groups of exposure: F 500 ppmyears Ž n s 54., 501]2500 ppm-years Ž n s 62., 2501]5000 ppm-years Ž n s 51. and ) 5000 ppmyears Ž n s 58.. For the whole group, the mean age and exposure were respectively 53.8" 8.7 years and 4909.8" 7431.8 ppm-years. The control group included 111 subjects hospitalized with non-cancer diagnoses and group-matched to the exposed workers for age, gender, race, smoking status and alcohol consumption. Results of mutant p21 or p53 proteins were stratified in quartiles of VC exposure, and crude Mantel]Haenzel odds ratios and 95% confidence intervals were calculated with the control group as reference ŽBrandt-Rauf et al., 1995; Li et al., 1998; Smith et al., 1998.. The crude odds ratios for the presence of the Asp13p21 protein increases from 8.48 Ž95% CI 3.01]23.89. for the group of exposure F 500 ppm-years to 21.73 ŽCI 8.76]53.94. for the group of highest exposure Ž) 5000 ppm-years.. The trend for increasing positivity with exposure is statistically significant Ž x 2 s 41.41, P- 0.00001.. When the unexposed controls were matched to the VC-exposed workers and odds ratios and 95% CI adjusted for age, alcohol consumption and smoking, the trend for increasing biomarker posi-

Table 1 Ki-ras and p53 gene analysis of vinyl chloride-related angiosarcomas of the liver and detection of mutated p21 and p53 proteins and anti-p53 antibodies in the serum from the same patients Ki-ras

p53

Tissue

ASL 1 ASL 2 ASL 3 ASL 4 ASL 5b ASL 6 a

Asp13p21 protein

Serum Asp13p21 protein

Tissue DNA

Serum

DNA codon 13 GGC ª GAC

Mutant p53 protein

Anti-p53

q q q q y y

q q q q y y

q q q q y y

ATCª TTC AGGª TGG ] ] CATª CTT nd

q q y y " y

ya qq y y q qq

Patient with known abnormal immunological response. Cell line established from an ASL. nd, not determined.

b

M.-J. Marion r Toxicology Letters 102]103 (1998) 603]607

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Table 2 Serum samples with anti-p53 antibodies Age Žin years.

Exposure before 1974 Žin years.

Asp13p21

Mutant p53

vWf ŽUrml.

Observations

59 49 68 66 56

5 16 23 23 14

q y y q y

q q q y y

1.05 1.77 1.45 1.61 1.70

Hepatitis B Steatosis Smoker None Smoker

vWf, von Willebrand factor.

tivity remains statistically significant ŽLi et al. Ž1998. and Li et al., unpublished results .. The crude odds ratios for the presence of the mutant p53 protein increases from 4.77 Ž95% CI 2.04]11.16. for the group of exposure F 500 ppm-years to 12.14 ŽCI 5.63]26.18. for the group of highest exposure Ž) 5000 ppm-years.. The trend for increasing positivity with exposure is also statistically significant Ž x 2 s 35.65, P 0.00001.. When the unexposed controls were matched to the VC-exposed workers, the trend for increasing biomarker positivity remains statistically significant ŽSmith et al., 1998.. Despite some bias due to the absence of true VC-exposure levels, the possible inaccuracy related to the confounding factors, and the use of hospitalized patients as controls, the statistical analysis for both markers shows a significant dose]response relationship between the serum markers positivity and the VC-exposure and supports our hypothesis that these mutations are induced by VC. These mutant proteins appear as markers of early biological effects. Are they also early tumor markers? For the exposed-workers without cancer, 72 and 89 samples were respectively positive for Asp13p21 and mutant p53 and 32 were positive for both markers. It is unlikely that 32 subjects will develop an ASL. However, five samples contained p53 antibodies, of whom three were also positive for the mutant protein ŽTable 2.. Four subjects exhibited no clinical sign of any VC-related disease whereas they exhibited an increased level of von Willebrand factor, a protein specifically synthesized by the endothelial cells, which is heavily increased in the blood of patients

with ASL and slightly but significantly increased for VC-exposed workers without cancer ŽFroment et al., 1991.. Thus a follow-up of this cohort is still necessary to assert the predictive value of these markers. Acknowledgements I would like to acknowledge all those who have contributed to this work and particularly Professor P. Brandt-Rauf, I. DeVivo, S. Smith and Y. Li. This work was supported in part by Association pour la Recherche sur le Cancer, Fondation de France, NIEHS ŽESO 5948. and Elf-Atochem. References Barbin, A., Bartsch, H., Lecomte, P., Radman, M., 1981. Studies on the miscoding properties of 1, N 6-ethenoadenine and 3, N 4-ethenocytosine, DNA reaction products of vinyl chloride metabolites, during in vitro DNA synthesis. Nucl. Acids Res. 9, 375]387. Barbin, A., Besson, F., Perrard, M.H., Bereziat, J.C., Kaldor, ´´ J., Michel, G., Bartsch, H., 1985. Induction of specific base-pair substitutions in E. coli trp A mutants by chloroethylene oxide, a carcinogenic vinyl chloride metabolite. Mutat. Res. 152, 147]156. Brandt-Rauf, P.W., Marion, M.J., DeVivo, I., 1995. Mutant p21 protein as a biomarker of chemical carcinogenesis in humans. In: Mendelsohn, M.L., Peeters, J.P., Normandy, M.J. ŽEds.., Biomarkers and Occupational Health Progress and Perspectives. Joseph Henry Press, Washington, DC, pp. 163]175. Creech, J.L., Jr., Johnson, M.N., 1974. Angiosarcoma of the liver in the manufacture of polyvinyl chloride. J. Occup. Med. 16, 150]151. DeVivo, I., Marion, M.J., Smith, S.J., Carney, W.P., BrandtRauf, P.W., 1994. Mutant c-Ki-ras p21 protein in chemical carcinogenesis in humans exposed to vinyl chloride. Cancer Causes Control 5, 273]278. Froment, O., Marion, M.J., Lepot, D., Contassot, J.C., Trepo,

M.-J. Marion r Toxicology Letters 102]103 (1998) 603]607 C., 1991. Immunoquantitation of von Willebrand factor Žfactor VIII-related antigen. in vinyl chloride exposed workers. Cancer Lett. 61, 201]206. Heldaas, S.S., Langard, S.L., Anderson, A., 1984. Incidence of cancer among vinyl chloride and polyvinyl chloride. Cancer Causes Control 5, 273]278. Hollstein, M., Marion, M.J., Lehman, T., Welsh, J., Harris, C.C., Martel-Planche, G., Kusters, I., Montesano, R., 1994. p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers. Carcinogenesis 15, 1]3. Li, Y., Asherova, M., Marion, M.J., Brandt-Rauf, P.W., 1998. Mutant oncoprotein biomarkers in chemical carcinogenesis. In: Mendelsohn, M.L., Mohr, L.C., Peeters, J.P. ŽEds.., Biomarkers: Medical and Workplace Applications. Joseph Henry Press, Washington, DC, pp. 345]353. Maltoni, C., Lefemine, G., 1974. Carcinogenicity bioassays of vinyl chloride. I } Research plan and early results. Environ. Res. 7, 387]405. Marion, M.J., Froment, O., Trepo, C., 1991. Activation of Ki-ras gene by point mutation in human liver angiosarcoma associated with vinyl chloride exposure. Mol. Carcinogen. 4, 450]454.

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Matsuda, T., Yagi, T., Kawanish, M., Matsui, S., Tabebe, H., 1995. Molecular analysis of mutations induced by 2-chloroacetaldehyde, the ultimate carcinogenic form of vinyl chloride in human cells using shuttle vectors. Carcinogenesis 16, 2389]2394. Przygodzki, R.M., Finkelstein, S.D., Keohavong, P., Zhu, D., Bakker, A., Swalsky, P.A., Soini, Y., Ishak, K.G., Bennett, W.P., 1997. Sporadic and Thorotrast-induced angiosarcomas of the liver manifest unfrequent and multiple point mutations in Ki-ras-2. Lab. Invest. 76, 153]159. Smith, S.J., Li, Y., Whitley, R., Marion, M.J., Partilo, S., Carney, W.P., Brandt-Rauf, P., 1998. Molecular epidemiology of p53 protein mutations in workers exposed to vinyl chloride. Am. J. Epidemiol. 147, 302]308. Trivers, G.E., Cawley, H.L., DeBenedetti, V.M.G., Prives, C., Hollstein, M., Marion, M.J., Bennett, W.P., Hoover, M.L., Tamburro, C.H., Harris, C.C., 1995. Anti-p53 antibodies in the serum of workers occupationally exposed to vinyl chloride. J. Natl. Cancer Inst. 87, 1400]1407. Viola, P.L., Bigotti, A., Caputo, A., 1971. Oncogenic response of rat skin, lungs and bones to vinyl chloride. Cancer Res. 31, 516]522.