Polymorphisms in glutathione S-transferases in French vinyl chloride workers.

The authors have recently demonstrated a significant gene-environment interaction between vinyl chloride exposure and polymorphisms in the DNA repair protein XRCC1 on the occurrence of mutant p53 biomarkers of vinyl chloride-induced genetic damage. The aim of this study was to examine the polymorphisms in the glutathione S-transferases (GSTs) as potential modifiers of this relationship, since these enzymes may be involved in the phase II metabolism of the reactive intermediates of vinyl chloride. A cohort of 211 French vinyl chloride workers was genotyped for common polymorphisms in GSTM1, GSTT1 and GSTP1. Although no independent, statistically significant effect of these polymorphisms on the occurrence of the mutant p53 biomarker was found, the null GSTM1 and null GSTT1 polymorphisms were found to interact with the XRCC 1 polymorphism to increase the occurrence of the biomarker such that, for example, workers with at least one variant XRCC1 allele who were null for both GSTM1 and GSTT1 had a significant odds ratio for the biomarker (OR =8.4, 95% CI = 1.3 54.0) compared with workers who were wild-type for all alleles, controlling for potential confounders including cumulative vinyl chloride exposure.

Establishment and characterization of a spontaneously immortalized myofibroblast cell line derived from a human liver angiosarcoma.

BACKGROUND/AIM: Fibrosis and/or cirrhosis are present in the precursor stages of most liver cancers. However, little is known about the reciprocal interactions of fibroblasts, mainly responsible for fibrosis, and the other liver cells. We report here the isolation of a new liver myofibroblast cell line from a human liver angiosarcoma and its characterization. METHODS: The cells were isolated by the explant technique and characterization was performed, on one hand, using immunohistochemical and ultrastructural analysis and, in the other hand, by determining their karyotype, ras and p53 status and their tumorigenic properties. RESULTS: To date, the cells have undergone approximately 170 population doublings and are still proliferating. Immunohistochemically, they were negative for desmin, smooth muscle myosin, cytokeratin 19 and von Willebrand factor, positive for vimentin and alpha-smooth muscle actin, with an important deposition of fibronectin around the cells. Ultrastructure showed particularly cytoplasmic microfilament bundles. Their chromosome number ranged from 38 to 168 with a bimodal population, near diploid and hypotetraploid. No mutations were found in codons 12, 13 or 61 of Ha-, Ki- and N-ras genes but a homozygous missense mutation in codon 179 (CAT-->CTT) was detected in the p53 gene. They were unable to form foci in soft agar or tumors in nude mice. CONCLUSIONS: Taken together, these results show that these cells, called BM 2.2.1, exhibited typical myofibroblast-like features. Although they contained a karyotype suggestive of tumoral cells and a homozygous mutated p53 gene, they were not tumorigenic. The nature of these cells and the abnormalities of the p53 gene and the karyotype, suggest that: i) they were a component of the tumor stroma, and ii) they could have been involved in angiosarcoma development. Thus, this cell line may be valuable for the study of cellular interactions in liver carcinogenesis.

Human hemangiosarcomas have a common polymorphism but no mutations in the connexin37 gene.

Gap junctional intercellular communication is often impaired in cancers, and the genes which encode the connexin gap junction proteins are considered to be tumor-suppressor genes. In this study, we analyzed the presence of mutations in the connexin 37 (Cx37) gene in 22 human hepatic angiosarcomas, 6 and 4 of which were associated with exposure to vinyl chloride and Thorotrast, respectively. The other 12 samples were from patients with no history of exposure to these 2 agents. In 9 samples, a proline (ACC) to serine (ACT) amino acid change in codon 319 was detected. However, DNA from non-tumorigenic tissue of the same patients also showed this amino acid change, suggesting that this is a polymorphism rather than a mutation. Subsequent analysis of 84 DNA samples from normal donors revealed the frequencies of Pro/Pro, Pro/Ser and Ser/Ser alleles to be 65.5%, 23.8% and 10.7%, respectively, while among the group of angiosarcoma patients the corresponding figures were 59.1%, 31.8% and 9. 1%, respectively. Thus, there was no correlation between the polymorphism at codon 319 and hepatic angiosarcoma occurrence. However, among the 6 cases of vinyl chloride-associated angiosarcoma, the percentages of the polymorphic alleles were 33.3%, 66.7% and 0%, respectively. While the number of samples was too small to allow us to conclude that the Ser319 allele in Cx37 predisposes to this rare type of human cancer, it may be noted that codon 319 is located at the cytoplasmic tail of Cx37, where most regulatory sequences reside, and that it could be a site of phosphorylation for some protein kinases, which may in turn affect the function of Cx37, including intercellular communication.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species.

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.

Common conformational effects in the p53 protein of vinyl chloride-induced mutations.

The tumor suppressor gene p53 has been identified as the most frequent site of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen, has been associated with specific A --> T transversions at codons 179, 249, and 255 of the p53 gene. The mutations result in amino acid substitutions of His --> Leu at residue 179, Arg --> Trp at residue 249, and He --> Phe at residue 255 in highly conserved regions of the DNA-binding core domain of the p53 protein. We previously used molecular dynamics calculations to demonstrate that the latter two mutants contain certain common regions that differ substantially in conformation from the wild-type structure. In order to determine whether these conformational changes are consistent for other p53 mutants, we have now used molecular dynamics to determine the structure of the DNA-binding core domain of the Leu 179 p53 mutant. The results indicate that the Leu 179 mutant differs substantially from the wild-type structure in certain discrete regions that are similar to those noted previously in the other p53 mutants. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure, but accessible in the mutant structure, and another region (residues 94-110) contains the epitope for the monoclonal antibody PAb1620, which is accessible in the wild-type structure, but concealed in the mutant structure. Immunologic analyses of tumor tissue known to contain this mutation confirmed these predicted conformational shifts in the mutant p53 protein.

Vinyl chloride-specific mutations in humans and animals.

Vinyl chloride is a potent hepatocarcinogen which reacts with DNA to generate etheno bases. In order to determine whether mutational patterns in target genes in vivo are characteristic of vinyl chloride and could be explained by the mutagenic properties of the etheno bases, human and rat liver tumours associated with exposure to vinyl chloride were analysed for point mutations in the ras and p53 genes. In this paper, we review these data and report our latest results on animal tumours. Two alterations were found which could be attributed to a direct effect of vinyl chloride: a GC-->AT transition which leads to a GGC-->GAC mutation at codon 13 of the Ki-ras gene in human liver angiosarcomas, and lesions at AT base pairs, mostly AT-->TA transversions, which lead to mutations in the p53 gene in human and rat angiosarcomas and to a CAA-->CTA mutation at codon 61 of the Ha-ras gene in rat hepatocellular carcinomas.

Molecular epidemiology of p53 protein mutations in workers exposed to vinyl chloride.

The production of mutations in cellular tumor suppressor genes such as p53 is involved in the development of many human cancers. These mutations result in the expression of mutant forms of the encoded p53 protein which can potentially serve as a biomarker for this carcinogenic process. Workers exposed to vinyl chloride who are at risk for the development of the sentinel neoplasm angiosarcoma of the liver represent a model population for the study of such a mutant p53 biomarker, since vinyl chloride is known to cause specific p53 mutations in persons with angiosarcoma of the liver. To determine the relation between vinyl chloride exposure and this p53 biomarker, the authors examined serum samples collected between 1987 and 1992 from a cohort of 225 French vinyl chloride workers and 111 unexposed controls (matched according to age, sex, race, smoking, and alcohol drinking) for the presence of mutant p53 protein, using an enzyme-linked immunosorbent assay. Stratification of the exposed workers by quartile of vinyl chloride exposure (in estimated ppm-years) yielded a statistically significant trend of increasing odds ratios for p53 biomarker seropositivity with increasing exposure. These results suggest that this serum biomarker for mutant p53 protein is related to vinyl chloride exposure and may be an early indicator of carcinogenic risk in exposed individuals.

Critical genes as early warning signs: example of vinyl chloride.

We have analysed liver angiosarcomas from individuals having been occupationally exposed to vinyl chloride (VC) to identify, in cancer-related genes, lesions which could be VC-specific. Two genetic alterations have been identified: the first one is a GGC --> GAT (Gly --> Asp, Asp13p21) mutation at codon 13 in the Ki-ras gene, found in five out of six tumors. The second one is an AT --> 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.

p53 gene mutation pattern in rat liver tumors induced by vinyl chloride.

Vinyl chloride (VC) induces angiosarcomas of the liver (ASL) and hepatocellular carcinomas (HCCs) in humans and rodents. We examined the presence of p53 gene mutations in ASL and HCC induced by VC in Sprague Dawley rats; 25 ASL and eight HCCs were analyzed for point mutations in exons 5-8, using PCR amplification, single-strand conformation polymorphism analysis, and direct DNA sequencing. Mutations were found in 11 (44%) of the ASL and in 1 HCC. A 12-base pair deletion was found in one tumor; all others were base pair substitutions. Nine of the point mutations were observed at A:T base pairs (5 A:T --> T:A; 2 A:T --> G:C, and 2 A:T --> C:G), and of three G:C --> A:T transitions, only one was at a CpG site. In ASL, four mutations were found in exon 5, two in exon 6, and six in exon 7; the base pair substitution found in one HCC was in exon 8. One ASL exhibited two point mutations, including a silent one. Two ASL exhibited the same mutation in codon 203 and two other samples in codon 253. Codon 235 was found to be mutated in three ASL. These data show that p53 is often mutated in ASL induced by VC in rats and, as observed in ASL in humans exposed to VC, the majority of the missense mutations involved A:T base pairs. The characteristic patterns of mutations found suggest that a common mechanism operates in VC-induced p53 mutagenesis in both species, and these mutations are consistent with the formation of DNA etheno adducts by VC in the liver. The A:T --> T:A transversion observed in the first nucleotide of codon 253 in two rat ASL is equivalent to the A:T --> T:A transversion characterized previously in codon 255 in one human ASL associated with VC exposure.

Conformational effects in the p53 protein of mutations induced during chemical carcinogenesis: molecular dynamic and immunologic analyses.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Vinyl chloride, a known human carcinogen that induces the rare sentinel neoplasm angiosarcoma of the liver, has been associated with specific A-->T transversions at the first base of codons 249 and 255 of the p53 gene. These mutations result in an Arg-->Trp amino acid substitution at residue 249 and an Ile-->Phe amino acid substitution at residue 255 in a highly conserved region in the DNA-binding core domain of the p53 protein. To determine the effects of these substitutions on the three-dimensional structure of the p53 protein, we have performed molecular dynamics calculations on this core domain of the wild-type and the Trp-249 and Phe-255 mutants to compute the average structures of each of the three forms. Comparisons of the computed average structures show that both mutants differ substantially from the wild-type structure in certain common, discrete regions. One of these regions (residues 204-217) contains the epitope for the monoclonal antibody PAb240, which is concealed in the wild-type structure but accessible in both mutant structures. In order to confirm this conformational shift, tumor tissue and serum from vinyl chloride-exposed individuals with angiosarcomas of the liver were examined by immunohistochemistry and enzyme-linked immunosorbent assay. Individuals with tumors that contained the p53 mutations were found to have detectable mutant p53 protein in their tumor tissue and serum, whereas individuals with tumors without mutations and normal controls did not.

Anti-p53 antibodies in sera of workers occupationally exposed to vinyl chloride.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is often mutated in a wide variety of cancers, including angiosarcoma of the liver (ASL). Anti-p53 antibodies have been detected in the sera of patients with leukemia, childhood lymphoma, or cancers such as those of the breast, lung, colon, esophagus, and liver (hepatocellular carcinoma). PURPOSE: The objective of this study was to determine the prevalence and time of appearance of serum anti-p53 antibodies during the pathogenesis of ASL associated with occupational exposure to vinyl chloride. METHODS: Enzyme-linked immunoassay (EIA) was used to detect anti-p53 antibodies in 148 serum samples from 92 individuals occupationally exposed (in France or in Kentucky) to vinyl chloride; 15 of these individuals (six from France and nine from Kentucky) had ASL. A subset of coded EIA-positive and EIA-negative sera was further analyzed for anti-p53 antibodies by immunoblotting and immunoprecipitation. Nucleotide sequence analysis of exons 5-8 of the p53 gene was conducted on ASL DNA from six patients. We tested sera from 31 men who had no occupational exposure to vinyl chloride; they made up the control group. Statistical analyses were done using the Kruskal-Wallis chi-squared approximation and the Wilcoxon two-sample test for normal approximation. All P values result from two-sided tests. RESULTS: Fourteen serum samples (from nine individuals) were positive in the EIA. Five of the 15 individuals with ASL were positive for anti-p53 antibodies by EIA, immunoblotting, and immunoprecipitation: one individual at 11.3 and 10.8 years before diagnosis, another at 4 months before and shortly after diagnosis, and three when diagnosed or shortly thereafter. Four of the 77 vinyl chloride-exposed workers without diagnosed ASL were positive for anti-p53 antibodies; two of the four had symptoms related to vinyl chloride toxicity. Tumors from three of the six vinyl chloride-exposed workers from which sufficient DNA for analysis was obtained had A:T to T:A missense mutations of the p53 gene. Anti-p53 antibodies were detected in two of these individuals. Among the control group, two of 15 serum samples from 15 lung cancer patients and zero of 15 serum samples from control subjects without cancer had anti-p53 antibodies as substantially lower levels than the nine (10%) of 92 vinyl chloride-exposed workers who were positive for anti-p53 antibodies. CONCLUSIONS AND IMPLICATIONS: Serum anti-p53 antibodies can predate clinical diagnosis of certain tumors, such as ASL, and may be useful in identifying individuals at high cancer risk, such as workers with occupational exposure to vinyl chloride.

The molecular epidemiology of growth signal transduction proteins.

Cellular proteins involved in growth signal transduction pathways represent potential molecular epidemiologic biomarkers for various disease processes of toxicologic interest, particularly cancer. In many instances, the proteins gain access to the extracellular environment and thus can be detected in easily accessible biological fluids such as serum or plasma. This paper reviews the experience with selected examples of such proteins, including growth factors, growth factor receptors, G proteins, and nuclear DNA-binding proteins, as serum or plasma biomarkers of disease.

Mutagenesis of ras proto-oncogenes in rat liver tumors induced by vinyl chloride.

Vinyl chloride is a DNA-damaging carcinogen which induces liver angiosarcomas in humans and animals. Activation of the Ki-ras 2 gene by a GC-->AT transition at the second base of codon 13 in human liver angiosarcomas associated with occupational exposure to vinyl chloride has been reported recently. In order to compare the molecular pathways of carcinogenesis in humans and animals, Sprague-Dawley rats were exposed to vinyl chloride and hepatic tumors, including two hepatocellular carcinomas and five liver angiosarcomas, were investigated for mutations at codons 12, 13 and 61 of the Ha-ras, Ki-ras and N-ras genes. High molecular weight DNA was amplified by the polymerase chain reaction and point mutations were analyzed by allele specific oligonucleotide hybridization, direct sequencing of polymerase chain reaction products and sequencing after cloning. None of the tumors exhibited a mutation in codons 12, 13 and 61 of the Ki-ras gene, nor in codons 12 of the Ha-ras gene or 61 of the N-ras gene. However, an activating AT-->TA transversion at base 2 of codon 61 of the Ha-ras gene was detected in the two hepatocellular carcinomas. Mutations involving codon 13 (GGC-->GAC) and codon 36 (ATA-->CTA) of the N-ras A gene were detected in two liver angiosarcomas, suggesting that the nature of the ras gene affected by a given carcinogen depends on host factors specific to cell types. Several additional base pair substitutions were found in exon 1 of the N-ras B and C sequences. NIH 3T3 transfection assays and Southern blot analysis of DNA from transformed NIH 3T3 cells confirmed the presence of a dominant activated N-ras gene. These results emphasize the differences in the molecular pathways leading to tumors in humans and rats and within a given species between different cell types.

Mutant c-Ki-ras p21 protein in chemical carcinogenesis in humans exposed to vinyl chloride.

Mutations in ras oncogenes and expression of their encoded p21 protein products are believed to play an important role in carcinogenesis in humans. Detection of mutant p21 proteins in serum may be a useful molecular epidemiologic biomarker with which to study this process, and workers with heavy exposure to vinyl chloride (VC) represent a model population for such study. We studied the occurrence of a specific ras mutation (Asp 13 c-Ki-ras) by oligonucleotide hybridization and the expression of the corresponding p21 protein in tumor tissue and serum by immunohistochemistry and immunoblotting with monoclonal antibodies in five individuals with heavy exposure to VC and resultant angiosarcomas of the liver (ASL). Four of five (80 percent) of the cases of ASL were found to contain the mutation and to express the corresponding mutant protein in their tumor tissue and serum. Serum expression of the mutant protein also was examined in nine VC-exposed workers with liver angiomas and 45 VC-exposed workers with no evidence of liver neoplasia; eight of nine (89 percent) of the former and 22 of 45 (49 percent) of the latter were also positive for the mutant p21 in their serum. However, serum immunoblotting results for 28 age-gender-race matched, unexposed controls were all negative. Stratification by years of VC exposure showed a significant linear trend (P < 10(-5)) for the occurrence of the serum mutant p21 protein with increasing duration of exposure. These results suggest that detection of serum mutant p21 protein can be a valid surrogate for ras gene expression at the tissue level.(ABSTRACT TRUNCATED AT 250 WORDS)

p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride-exposed factory workers.

Mutations in the p53 tumor suppressor gene are commonly found in the major human cancers and the mutational spectrum in some cancer types is consistent with the genotoxic effects of the associated environmental risk factors. Thus far there is little information on p53 mutations in cancers of factory workers with a history of carcinogen exposure in the workplace. Occupational exposure to vinyl chloride causes liver angiosarcomas (ASL) and also increases the risk of several other cancers. Loss of p53 function in osteo- and fibrosarcomas can occur by two different mechanisms, p53 mutation and amplification of the MDM2 gene. We examined tumors from five vinyl chloride-exposed patients, four with ASL and one with hepatocellular carcinoma (HCC), for evidence of MDM2 proto-oncogene amplification or p53 mutation in exons 5-8. Amplification of MDM2 was not found, but in two of the angiosarcomas an A:T to T:A missense mutation was detected. p53 sequence analysis of vinyl chloride associated cancers may provide valuable information on the relationship between carcinogen exposure and DNA damage in cancer-related genes.

Skeletal dysplasia in perinatal lethal osteogenesis imperfecta. A complex disorder of endochondral and intramembranous ossification.

Osteogenesis imperfecta (OI) Type II is a rare heritable disorder of bone matrix that results in catastrophic congenital skeletal dysplasia. Two cases of OI Type II had symmetric rhizomelic skeletal dysplasia apparent on ultrasound at 16 and 20 weeks' gestation. Histologic and histochemical studies performed on skeletal tissue from fetal autopsies showed the following: (1) abnormal growth plate tissue characterized by failure of formation of primary bony spongiosa; (2) persistence of calcified cartilage bars in the diaphysis; (3) metaphyseal microfractures; (4) abundant cartilaginous fracture callus; (5) absence of bony callus; (6) failure of formation of intramembranous cortical diaphyseal bone; (7) angulation of long bones in portions of the metadiaphyses bordered by fracture callus; and (8) mechanical failure of the perichondral ring of LaCroix with a normal fibrous ossification groove of Ranvier. These findings suggest that skeletal dysplasia in OI Type II results from the action of muscular forces on a skeleton weakened by a complex disorder of endochondral and intramembranous ossification. The paucity of primary metaphyseal trabeculae and subperiosteal cortical bone leads to pathologic fractures of the immature fiber bone and an imperfect attempt at fracture repair. Angulation and shortening of long bones occurs between numerous sites of focal endochondral fracture callus. Mechanical failure of the fibrous perichondral ring leads to further collapse and shortening without obvious functional impairment of the fibrous ossification groove. Perinatal lethal OI provides insight into how a molecular disorder predominantly of Type I collagen metabolism results in pathology of numerous tissues, leading to severe skeletal dysplasia without primarily affecting chondrogenesis.

Immunoquantitation of von Willebrand factor (factor VIII-related antigen) in vinyl chloride exposed workers.

Angiosarcoma of the liver is a rare malignant tumor which has been associated with occupational exposure to vinyl chloride (VC). We have determined by ELISA the level of von Willebrand factor (vWf) in the serums of 107 VC-exposed workers, active or retired, and of 133 blood donors used as controls. The vWf level was slightly but significantly higher in the VC-exposed group than in the control group (P = 0.035). Seventeen VC-exposed workers exhibited a raised level of vWf, with no biochemical sign of hepatic disturbance, nor any evidence of illness; only one of them exhibited elevated alkaline phosphatase and gamma-glutamyl transpeptidase values. The vWf serum level of 3 patients with hepatic angiosarcoma associated to VC-exposure was markedly elevated. These increased levels of vWf in VC-exposed workers most likely reflect an increased activity of liver endothelial cells; whether an elevated level of vWf could be associated with increased risk of developing liver angiosarcoma remains to be determined.