Genetic Polymorphisms, Messenger RNA Expression of p53, p21, and CCND1, and Possible Links with Chromosomal Aberrations in Chinese Vinyl Chloride–Exposed Workers

Vinyl chloride monomer (CH₂ = CHCl, VCM) is a widely used gas in industries that produce polyvinyl chloride. Although the relationship between VCM exposure and angiosarcoma/hepatocellular cancer has been established (1, 2), the mechanism behind it remains unclear. At present, the coherent view about the mechanism is that the two metabolites of VCM (chloroethylene oxide and chloroacetaldehyde) interact with DNA to form DNA adducts and induce errors during DNA and RNA synthesis (3). Previous epidemiologic studies have shown that VCM exposure is associated with increased genotoxicity in humans. Chromosomal aberrations, micronuclei, sister chromatid exchanges, and DNA strand breaks (the comet assay) have been observed in lymphocytes of individuals occupationally exposed to VCM (4-6).

Among the effects of the biomarkers mentioned above, the frequency of micronuclei in human cells has become one of the standard cytogenetic indexes for genetic toxicology testing. The cytokinesis-block micronucleus (CBMN) assay is the preferred method for measuring micronuclei in cultured human cells because scoring is specifically restricted to once-divided cells. Because micronucleus formation requires nuclear division, the scoring of those cells that have completed nuclear division is a prerequisite for the correct interpretation of the observed micronuclei frequencies. This is achieved by scoring micronuclei in binucleated cells using the CBMN technique (7, 8).

Earlier epidemiologic studies showed that exposure to VCM has been associated with hepatoma and angiosarcoma of the liver. However, the majority of VCM-exposed workers do not develop tumors. This susceptibility is modulated at least in part by polymorphisms in genes encoding metabolic enzymes, DNA repair proteins, and cell cycle control proteins. Many studies have investigated the effect of genetic polymorphisms of genes involved in metabolism (CYP2E1, GST, mEH, ALDH2) and DNA repair (XRCC1, XPD; refs. 6, 9-15). To the best of our knowledge, however, no studies have been conducted on the effect of genetic polymorphisms of genes involved in cell cycle control.

p53, p21, and CCND1 are important genes involved in the G₁-S checkpoint. In the presence of DNA damage after exposure to a carcinogen, up-regulation of p53, and subsequently, p21 could delay progression past the G₁ restriction point. CCND1 acts as an active switch that regulates the transition of the cell cycle from the G₁ phase to the S phase (16). Mutations in p53, p21, or CCND1 may lead to the change of gene expression and loss of this homeostatic control and thereby induce human carcinogenesis.

p53 is critical for cell cycle control and DNA repair, and is known to contain a variety of polymorphisms. Both codon 72 in exon 4, 16-bp duplication in intron 3, and a G → A transition in intron 6 polymorphism are common and may be related with some cancers (17). p21 is one of the notable effectors of p53, and it is a general inhibitor of cyclin-dependent kinases. It functions to negatively control the cell cycle (18, 19). p53 up-regulates p21 expression in response to DNA damage, leading to cell cycle arrest at the G₁ checkpoint (19). Polymorphisms of p21 have been speculated to change its function and the high-risk genotypes have been reported to be associated with different cancers (20-24). The two most studied polymorphisms of p21 were Ser > Arg at codon 31 and a G → T transition at the 70th base in exon 3. CCND1 acts as a positive function in cell cycle control and it is known that it has a single base polymorphism (G870A) in exon 4 that increases the frequency of alternate splicing (25) and is involved in various types of human cancer (26). In this study, we genotyped for cell cycle control genes including p53, p21, and CCND1, and investigated the relationships between the genotype and the frequencies of CBMN in peripheral blood lymphocyte among VCM-exposed workers.

Following genotoxic stress, the p53 protein is activated by specific phosphorylation events that lead to increased levels of p53-responsive target genes—p21—resulting in the cell cycle being interrupted and the inability of damaged DNA to be replicated. Besides gene polymorphism, VCM exposure is another factor influencing the mRNA, protein expression of the cell cycle control gene, and the results of DNA repair. Overexpression of p53 and p21 proteins can be found in the plasma of VCM workers and a significant dose-response relationship exists between plasma oncoprotein expression and VCM exposure (27), but whether this overexpression is due to p53 and p21 mRNA expression remains unclear. In addition, there is no report about the relationship between CCND1 mRNA expression and VCM exposure. In this study, we detected the alteration in mRNA level of p53, p21, and CCND1 among VCM-exposed and nonexposed workers.

Workers employed in a VCM polymerization plant in China were studied. Prior to the study, written informed consent was obtained from each subject and a questionnaire was used to determine the lifestyle of each subject, i.e., smoking and alcohol habits, medication history, and occupational history. Subjects exposed to VCM for longer than 1 year were selected for the polymorphism study if the following criteria were met: detailed questionnaires had been completed, the CBMN test results and a blood sample had been provided, and PCR-restriction fragment length polymorphism for all the studied genes was completed successfully. A total of 183 workers met these criteria. This study of p53, p21, and CCND1 mRNA expression was done with 77 occupationally exposed workers from the same VCM polymerization plant and 43 unexposed controls from the same city but without a history of exposure to VCM or other toxicants.

The level of VCM was measured for different work sites of the plant using gas chromatography. Because the VCM plant had kept VCM air concentration data for different work sites from the beginning of its establishment, we were able to estimate the cumulative exposure dose of each worker with a relatively high level of precision. The cumulative exposure dose was calculated according to an equation as described previously (6). The VCM-exposed subjects then were divided into high-exposure and low-exposure groups according to the median dose.

The CBMN assay was done according to standard methods as described previously (28, 29). Aliquots of 0.5 mL of heparinized whole blood were cultured in 4.5 mL of medium. Forty-four hours after phytohemagglutinin stimulation, cytokinesis was blocked with 6 μg/mL of cytochalasin-B (Sigma), followed 28 h later with the harvesting of lymphocytes and fixation with methanol and acetic acid at a 4:1 ratio before being transferred to slides. For each subject, 1,000 binucleated lymphocytes with well-preserved cytoplasms were scored blindly by the same reader.

p53, p21, and CCND1 genotypes were determined using genomic DNA isolated from peripheral blood lymphocytes and PCR-restriction fragment length polymorphism assays previously described (30-33).

Total RNA was isolated from the lymphocytes and quantified by measurement of the absorbance at 260 nm; purity was assessed from the 260/280 nm absorbance ratio. Real-time PCR was done using the one-step SuperScript kit (Life Technologies) according to the manufacturer's instructions. The quantity of p53, p21, and CCND1 gene mRNA expression was detected by real-time PCR (SYBR Green I) with PRISM ABI 7900HT Sequence Detection System (Applied Biosystem). All the primers were self-designed using Primer Premier 5 software. The sequences and lengths of the amplified fragments are listed in Table 1.

Real-time PCR was done using 50 ng of cDNA, 0.25 μL of each primer (10 umol/L), and 2× ABsolute QPCR SYBR Green Mix (including 500 nmol/L ROX; Abgene) in a 10 μL reaction volume. The PCR program was a 15-min activation step at 95°C, followed by 40 cycles of 95°C for 15 s, and finally, at 60°C for 1 min. Expression of the housekeeping gene glyceraldehyde 3-phosphoric acid dehydrogenase (GAPDH) was used for normalization of p53, p21, and CCND1 mRNA to enable cross-comparisons among the samples. Every sample was done using three parallels. The results were analyzed by PRISM ABI 7900HT Sequence Detection System (Applied Biosystem). The threshold cycle (C_t) values for the triplicate reactions were averaged and the average GAPDH C_t value for each sample was subtracted from the average C_t value of interest to obtain a normalized C_t value. The mRNA expression of objective genes was shown by 2^−ΔCt value.

We assess the statistical significance of tests for the Hardy-Weinberg equilibrium and linkage disequilibrium analysis using the method described by Shi and He (34). PHASE software (version 2.0.2) was used to obtain maximum-likelihood estimates of the p53 haplotype frequencies.

The SAS software package (version 9.1) was used for the other statistical analysis. The influence of genotype, age, gender, cumulative exposure dose, alcohol consumption, and smoking status on the frequencies of micronuclei were determined using univariate and multiple Poisson regression analyses. All possible two-way interactions among the above factors were tested. Frequency ratio (FR), its confidence interval (CI), and the corresponding P values were reported. For categorical variables, the FR indicates the proportional increase of the micronucleus frequency in the study group; for example, a FR of 1.21 for females versus males means a 21% increase of micronucleus frequency in females (35). In order not to ignore important risk factors in such an exploratory multivariate analysis, the test level was relaxed to 0.10 (as the default test level of stepwise linear regression model in SAS package is 0.15, not the usual 0.05). For the sake of consistency, the CIs of the estimations were calculated with a degree of confidence to 90%.

In studying p53, p21, and CCND1 mRNA expression, the demographic characteristics of the exposed and nonexposed study groups were compared using a t test for age and χ² tests a for gender as well as smoking and drinking habits. The median and interquartile range of p53, p21, and CCND1 mRNA expression of both exposed and nonexposed groups were calculated. In order to adjust for the influence of demographic characteristics, we did a multiple factor analysis using robust multiple regression (due to the non-normal distribution of the gene expression data), with PROC ROBUSTREG (S estimation)⁵

The overall genotype and M allele (variant allele) frequencies are shown in Table 2. No p53 intron 3 mutant homozygous genotype was detected, whereas the frequency of intron 6 mutant homozygous genotype was low (0.01). M allele frequencies of p53 Arg72Pro, p21 Arg31Ser, and CCND1 A870G were close to the data obtained from the single nucleotide polymorphism database of the National Center for Biotechnology Information (0.490, 0.386, and 0.483, respectively). But the M allele frequencies of other sites did not correspond to the database (0.500, 0.187, and 0.324 for p53 intron 3, p53 intron 6, and p21 exon 3, respectively). The FR estimated by Poisson regression analysis showed a significant increase of micronuclei in subjects with p53 Arg72Pro heterozygous genotypes or p53 intron 6 mutant homozygous, and/or heterozygous compared with their wild-type homozygous counterparts (P < 0.05). No detectable influence of the p21 Arg31Ser, p21 exon 3, and CCND1 A870G polymorphisms on micronuclei were observed in this study (P > 0.05). The distribution of the M alleles in all subjects was in agreement with the Hardy-Weinberg equilibrium; the only deviation was for p21 Arg31Ser (P = 0.009).

The female subjects had higher mean micronuclei frequencies than the male subjects (4.06 versus 3.43, respectively; P < 0.05). Subjects older than 35 years had higher mean micronuclei frequencies than their younger counterparts (4.21 versus 3.26; P = 0.01). No significant difference was observed in the mean micronuclei frequencies between smokers and nonsmokers, nor between habitual and nonhabitual drinkers. The high-exposure subgroup had higher micronuclei frequencies than the low-exposure subgroup (3.82 versus 3.52), however, the difference did not seem to be statistically significant either (P > 0.05; Table 3). In order to explore the relationship between the cumulative dose of VCM and micronuclei frequency, we next divided all the subjects into four subgroups by median and quartiles of the cumulative dose. We found that the micronuclei frequency increased in order from the lowest dose subgroup to the highest dose subgroup, from 3.49 ± 2.358 (n = 47), to 3.54 ± 2.062 (n = 48), 3.78 ± 2.745 (n = 51), and 3.86 ± 2.238 (n = 37), respectively. The difference between these subgroups, however, was not statistically significant (P > 0.05).

Because the frequencies of the variant homozygotes of p53 intron 3 and intron 6 were too small, we combined the heterozygote and variant homozygote into one group. The results of multiple Poisson regression, taking into account the effects of genetic polymorphisms and demographic and habitual factors, are summarized in Table 4. This analysis confirmed the increase in micronucleus frequency with gender and age (P = 0.0381 and P = 0.0017, respectively). Subjects with a p53 intron 6 variance showed a high micronucleus frequency compared with their wild-type homozygous counterparts (P = 0.0814). Other factors did not show any association with the level of micronuclei frequency.

To examine the linkage among the three loci (exon 4, intron 3, and intron 6), we did linkage disequilibrium analyses. The D′ value of the three loci of p53 were 0.997 (exon 4 with intron 3), 0.814 (exon 4 with intron 6), and 0.857 (intron 3 with intron 6). For all subjects, a total of 11 haplotype pairs were detected by the PHASE 2.0.2 software. Among these haplotype pairs, values more than three were named hap1 to hap5, and the remaining were combined into one group (named “others”), due to small sample sizes. FRs associated with various p53 haplotype pairs in all study subjects are presented in Table 5. Compared with individuals with wild-type hap1, the FR was 1.44 (95% CI, 1.02-1.99) and 1.71 (95% CI, 1.00-2.73) for the individuals with hap4 and hap5, respectively, and these differences were significant (P = 0.0322 and P = 0.0350, respectively). Multiple Poisson regression showed that FR adjusted by age, gender, and cumulative exposure dose increased slightly for the individuals with hap4 and hap5 (FR, 1.48; 95% CI, 1.05-2.06; and FR, 1.82; 95% CI, 1.06-2.92, respectively) compared with individuals with hap1 (P = 0.0233 and P = 0.0208, respectively).

The two groups in the mRNA expression study matched on the basis of gender as well as smoking and drinking habits, but the VCM-exposed group was significantly younger than the unexposed group (35.53 ± 5.57 versus 45.41 ± 5.97; P < 0.05). Robust multiple regression indicated that p53 mRNA expression of VCM-exposed workers was significantly lower than that of the nonexposed group (P < 0.001), but p21 mRNA expression in VCM-exposed workers was significantly higher than that of nonexposed workers(P < 0.001; Table 6). The difference of CCND1 mRNA expression between these two groups was not significant (P = 0.520).

Micronuclei are chromosomes or chromosomal fragments that were not incorporated into the main nucleus during cell division. The induction of micronuclei represents sensitive cytogenetic end points for the detection of genotoxic activity of environmental mutagens and carcinogens and the increased frequency of micronuclei has been observed in lymphocytes of individuals occupationally exposed to VCM (4). The CBMN assay is the preferred method for measuring micronuclei in cultured human cells, so it was employed in the present study and the frequency of micronuclei in lymphocytes was used to assess the genetic effect of occupational exposure to VCM. Our data also showed that the frequencies of the micronuclei were significantly higher in the VCM-exposed group than in the unexposed control group (to be published in a subsequent article). To investigate whether the DNA damage was associated with the polymorphisms of p53, p21, and CCND1, we evaluated the micronuclei frequency distributions of p53 (exon 4, intron 3, intron 6), p21 (Arg31Ser, exon 3), and CCND1 (A870G) polymorphisms in VCM-exposed workers. The results obtained suggest that only the p53 intron 6 polymorphism is involved in the modulation of the level of micronuclei.

Although p53 does not actively repair DNA, it is critical to the DNA repair process because its expression regulates cell cycle checkpoints that can pause the cell cycle to allow for DNA repair. One of the notable effectors of p53 is p21, which is a general inhibitor of cyclin-dependent kinases. p53 regulated p21 expression in response to DNA damage leading to cell cycle arrest at the G₁ checkpoint (18). CCND1 acts as an active switch that regulates the transition of the cell cycle from the G₁ phase to the S phase (16), hence, the polymorphisms and expression of p53, p21, and CCND1 might modify the level of DNA damage in VCM-exposed workers.

Genetic polymorphisms are common in the general population. Some polymorphisms are located within the coding region of a gene, whereas others are found in introns. Although introns were originally believed to be nonfunctional because they do not code for proteins, it has been suggested that some of these sequences do indeed have relevance (36). Mutations in intron sequences may initiate aberrant pre-mRNA splicing, producing an mRNA that may be translated into a defective protein (37). Furthermore, intronic polymorphisms may influence coding region mutations that increase the likelihood of a deleterious phenotype (38). Our data suggest that G > A polymorphisms at intron 6 of p53 are associated with a higher level of micronuclei frequency. The micronuclei frequencies in subjects with mutant homozygous and/or heterozygous alleles is higher than their wild-type homozygous counterparts. This finding suggests that the intron 6 M alleles might exert a functional effect. For the p53 16 bp duplication allele at intron 3, however, we did not observe any positive results. Several studies reported functional differences in genotypes at intron 3 and intron 6 polymorphisms of p53 gene (30, 36, 39, 40). One study reported a lower repair capacity of lymphoblastoid cell lines that harbor less frequent alleles of 16 bp duplication at intron 3 and a G > A polymorphism at intron 6 (30). Recently, reduced levels of p53 mRNA have been proposed to be associated with the p53 16 bp duplication allele at intron 3 (41). Overall, further larger-scale studies need to be conducted to unveil any regulatory role of these intronic sequences.

Linkage disequilibrium analysis of p53 found that the three polymorphisms of p53 (exon 4, intron 3, intron 6) are in strong linkage disequilibrium, which agreed with another earlier study by Sjalander et al. (42). Several studies have shown that the haplotypes composed of variants of multiple single nucleotide polymorphisms may be a more appropriate tool for assessing host-environment disease associations compared with individual single nucleotide polymorphisms (43, 44). Therefore, we analyzed the haplotype of exon 4, intron 3, and intron 6 of the p53 gene. With respect to haplotype pairs, MWW/WWW was the most prevalent haplotype (46.45%), whereas WWW/WWW haplotype pairs appeared 27.87% of the time. Wu et al. (30) suggested that the p53 minor haplotypes are associated with risk of lung cancer in Caucasians, with the highest risk found in individuals with the WMM haplotype. Consistent with this previous finding, we saw that the minor haplotype pairs (MMM/WWW and MWM/WWW) were associated with elevated risk of chromosome damage, with the highest micronuclei frequency found in individuals with the MWM/WWW haplotype. Such a statistically significant association may be attributable to changes in p53 function because the DNA repair capacity of mutant alleles was lower than that of wild-type alleles. Wu et al. (30) additionally measured the DNA repair capacity in 22 lymphoblastoid cell lines to determine the functional effects of the three polymorphisms of p53, and found that lymphoblastoid cell lines with all wild-type alleles at the three loci had statistically significant, higher DNA repair capacity than cell lines with at least one variant allele at all three loci.

Besides the polymorphism at intron 6, our data also indicated that the higher levels of micronuclei were associated with age and gender. Through analysis of a population sample from several biomonitoring studies done over the last few decades in 12 Italian laboratories, Bolognesi et al. (45) showed that the frequency of the mean standardized micronuclei values rose with increasing age. Ishikawa et al. (46) indicated that subjects >40 years of age had higher micronuclei frequencies than younger subjects (≤40 years). Our study also indicated that subjects >35 years of age had higher micronuclei frequencies than younger subjects (≤35 years). Another important finding of this analysis is the observation that women occupationally exposed to VCM are at higher risk for micronuclei frequency induction than occupationally exposed males, which confirms the work of Kirsch-Volders et al. (35). Together, the finding of statistically significant effects of age and gender on micronuclei frequency strongly suggest that human VCM genotoxic effect studies based on the cytogenetic markers of micronuclei need to take into account not only the exposure factors but also individual demographic factors.

Generally, DNA damage after exposure to a carcinogen leads to an up-regulation of p53, and subsequently, an increase in p21 mRNA and protein in cell cycle arrest at the border between the G₁ and S phases (47). Therefore, besides gene polymorphism, VCM exposure that may induce DNA damage could also increase the expression of p53 and p21 genes. A previous study has shown that p21 and p53 protein overexpression can be found in the plasma of VCM-exposed workers (27). In our study, p53 mRNA expression in VCM-exposed workers significantly decreased, but p21 mRNA expression significantly increased. The significant induction of p21 mRNA expression in VCM-exposed workers indicates that the p53 pathway is functional in VCM exposure. It is possible that this pathway involves an association of p53 with damaged DNA, triggering the accumulation of p53. In normal cells, p53 is rapidly degraded; in some circumstances, degradation occurs via the ubiquitin-dependent proteolytic pathway. Because inhibitors of cellular transcription and translation do not block p53 accumulation after DNA damage (48), p53 accumulation probably reflects a reduction in the rate of p53 degradation. A tight association with DNA could lead to reduced degradation of p53 through changes in p53 conformation, modification, association with other proteins, or compartmentalization within the nucleus. Subsequent DNA repair could lead to the release of p53 from damaged DNA and restoration of normal degradation processes. Therefore, the possible explanation for the discrepancy might be that a reduction in the rate of p53 degradation induced p53 accumulation and triggered p21 mRNA expression in turn. But the reason for the reduction of p53 mRNA expression is unknown. Our results indicate that accumulated p53 and p21 proteins have been implicated in the repair of DNA damage induced by VCM exposure. The p21 protein accumulates through increasing p21 mRNA expression, although p53 protein accumulates by reducing the rate of degradation.

In summary, we found for the first time that G > A polymorphism at intron 6 of p53 may contribute to the level of DNA damage in occupational exposure to VCM in a Chinese population. This finding, once verified by large studies, will have important implications in the prevention of tumors in susceptible workers. The strengths of this study include the homogeneous ethnic backgrounds of the subjects, and the well-documented exposure history to VCM. However, joint action between genetic polymorphisms and environmental exposure is complicated and small studies like the present study do not have enough statistical power to detect gene-environment interactions. Thus, a more comprehensive, larger-scale study is needed to further explore the effects of gene-environment interaction.

No potential conflicts of interest were disclosed.

We thank physicians Shangjian Chai and Jun Li for their help for physical examination of the workers and data collection of VCM exposure.