Ainsley Weston

Distribution of HER2V655 genotypes in breast cancer cases and controls in the United States

The minor variant frequency of a HER2 polymorphism (HER2(V655)) has been determined for 471 United States women enrolled in a multiracial case-control study. Allelic frequencies varied significantly by race. Genotypic distributions showed no excess breast cancer risk associated with inheritance of HER2(V655) either as carriers (OR=1.2, 95% CI=0.8-1.9), heterozygotes (OR=1.2, 95% CI=0.8-1.9), or homozygotes (OR=1.4, 95% CI=0.4-4.2). Nor was there a significant association when each racial group was considered separately. The current study suggests the HER2(V655) allele is not a breast cancer risk factor for Caucasians, African-Americans, or Latinas.

Association between GSTM1*0 and squamous dysplasia of the esophagus in the high risk region of Linxian, China.

Individuals with specific phase I and phase II enzyme polymorphisms may be at increased risk for squamous cell carcinoma of the esophagus. However, to our knowledge there has been only one previous report that evaluates a potential role for these polymorphisms in increasing risk for preneoplastic squamous lesions of the esophagus. To explore this further, we examined polymorphisms in CYP1A1, CYP2E1, GSTM1 and GSTT1, both independently and in combination, for potential associations with the risk of biopsy-proven squamous dysplasia of the esophagus in asymptomatic adults from Linxian, a high risk region in China. Cases consisted of 56 individuals from an esophageal cancer screening study with an endoscopic biopsy diagnosis of mild or moderate squamous dysplasia. Each case was matched on age (+/- 1 year) and gender to a control. Controls were defined as screening study participants with an endoscopic biopsy diagnosis of normal mucosa or esophagitis. DNA was extracted from frozen cell samples obtained by cytologic balloon examination and genotyped using standard methods. Individuals who were GSTM1 null (homozygous for GSTM1*0) were found to have a tendency for an increased risk of esophageal squamous dysplasia (odds ratio=2.6, 95% CI, 0.9-7.4). No excess risks were observed for inheritance of other putative at risk genotypes CYP1A1*2B, CYP2E1*6 or GSTT1*0. The risk associated with the inheritance of combined genotypes was not significantly different than the risk estimates from the univariate analysis. These results are consistent with the notion that exposure to environmental carcinogens that are detoxified by GSTM1, such as polycyclic aromatic hydrocarbons, may contribute to the etiology of esophageal cancer in Linxian.

DNA sequence variants of p53: cancer and aging.

To the Editor: n np53 has a critical role in cell-cycle control. As such, it has been identified as an important target in human carcinogenesis. However, since human p53 was cloned, ⩾10 DNA-sequence polymorphisms have been identified (Matlashewski et al. 1987; Weston and Godbold 1997). The codon 72 polymorphism (arginine/proline: G/C), the first to be described, has been the subject of ⩾31 epidemiological case-control studies that have explored a potential association with cancer (Olschwang et al. 1991; Weston et al. 1992, 1994, 1997; Zhang et al. 1992; Kawajiri et al. 1993; Birgander et al. 1995, 1996b; Jin et al. 1995; Sjalander et al. 1995a, 1996a; Wu et al. 1995; Murata et al. 1996; To-Figueras et al. 1996; Golovleva et al. 1997; Weston and Godbold 1997; Yung et al. 1997; Hayes et al. 1998; Helland et al. 1998; Hildesheim et al. 1998; Josefsson et al. 1998; Lanham et al. 1998; Minaguchi et al. 1998; Rosenthal et al. 1998; Storey et al. 1998; Tagawa et al. 1998; Wang-Gohrke et al. 1998). Although they err on the side of caution by citing Weston and Godbold (1997), Bonafe et al. (1999, p. 293), referring to the codon 72 polymorphism, state that “overall, the available data in the literature suggest that p53 variants may be considered as risk factors for some of the major neoplastic diseases in humans, such as lung, colorectal, breast and cervical cancer and are expected to affect survival.” With respect to codon 72, we contend that this is probably not the case. In the available data, lung cancer is the subject of eight studies (Weston et al. 1992, 1994; Kawajiri et al. 1993; Birgander et al. 1995; Jin et al. 1995; Murata et al. 1996; To-Figueras et al. 1996; Tagawa et al. 1998). Three studies claim a statistically significant association: in one study, a subset analysis suggested a relationship in lung cancer cases diagnosed at age <53 years (Jin et al. 1995); in the other two, the allelic frequencies were almost identical (and the difference was not significant) between cases and controls (Kawajiri et al. [1993] observed a proline-allele frequency of .35 for controls and .36 for cases [n = 347 and 328, respectively]). Murata et al. (1996) observed a proline-allele frequency of .40 for controls and .35 for cases (n = 152 and 191, respectively), but associations of cancer risk were claimed on the basis of higher numbers of homozygotes (Kawajiri et al. 1993; Murata et al. 1996). One study (Kawajiri et al. 1993) implicated proline; the other (Murata et al. 1996), arginine. n nMost recently, the relationship between cervical cancer, human papillomavirus (HPV) infection, and inheritance of the arginine allele has received considerable attention. The initial study, cited by Bonafe et al. (1999), showed a high degree of correlation between inheritance of the arginine allele and cervical cancer risk when HPV infection was present but not when it was absent (Storey et al. 1998). There are now eight studies of cervical cancer, but only one shows any association and seven are null (Hayes et al. 1998; Helland et al. 1998; Hildesheim et al. 1998; Josefsson et al. 1998; Lanham et al. 1998; Minaguchi et al. 1998; Rosenthal et al. 1998). There are five breast cancer studies (Kawajiri et al. 1993; Sjalander et al. 1996a; Weston et al. 1997; Helland et al. 1998; Wang-Gohrke et al. 1998); only one reached significance. Of the other published studies, a positive association has been claimed in 0/2 for nasopharyngeal cancer (Birgander et al. 1996b; Golovleva et al. 1997; Yung et al. 1997), 0/4 for colon cancer (Olschwang et al. 1991; Kawajiri et al. 1993; Jin et al. 1995; Sjalander et al. 1995a), 0/2 for bladder/urologic cancer (Kawajiri et al. 1993; Wu et al. 1995), 0/1 for acute myelogenous leukemia (Zhang et al. 1992), and 1/1 for stomach cancer (Kawajiri et al. 1993). Again, for stomach cancer, the allelic frequencies were not different for cases and controls, and an association of cancer risk with the arginine variant was claimed on the basis of higher numbers of arginine homozygotes in cases (Kawajiri et al. 1993; this report used the same control group noted above, and the proline-allele frequency in stomach cancer was .28 [n=140]). n nWe contend, therefore, that the balance of the results of human molecular epidemiological association studies suggests that codon 72 allelism does not have an impact on human cancer risk. Specifically, at most, only 6 of 31 positive associations have been reported, and none have received consistent support in attempts to replicate them (Kawajiri et al. 1993; Jin et al. 1995; Murata et al. 1996; Sjalander et al. 1996a; Storey et al. 1998). Moreover, there is no obvious, plausible biological basis for an association of cancer risk with the codon 72 polymorphism. The impact of proline on the tertiary structure of a protein is disruption of α-helices. A proline residue resides at the monomeric position codon 71; therefore, at codon 72 there is no obvious consequence to proline. n nResults were reported for p53, codon 72, genotype frequencies in healthy Italian centenarians and younger controls (Bonafe et al. 1999). The expectation was of allelic winnowing in the case of a codon 72 variant associated with cancer susceptibility and subsequent survival. The frequency of proline-allele carriers was .539 in controls and .506 in centenarians, 3.3% lower (and the corresponding allelic frequency was 2.7% lower, but the difference was not significant). We conjecture that their comparison of centenarians (n=176) with younger controls (n=204) is too crude to detect the predicted allelic winnowing (Bonafe et al. 1999). First, the cause of death from cancer in the Italian population is ∼23% (WHO 1987; Lopez 1990). Second, even though we do not agree that there is good evidence for an association between inheritance at codon 72 and cancer susceptibility, because of the molecular epidemiological data reviewed above, let us assume that the proline variant carries a relative risk of 1.5–2.0 (Kawajiri et al. 1993). On the basis of this assumption we might expect a reduction of 3.0%–4.8% in proline-allele carriers as the population ages (table 1 shows a simulated 2×2 table for a standard population of 1,000 persons, given a relative risk of 1.5 and a cancer death rate of 23% [WHO 1987; Lopez 1990]). In the letter by Bonafe et al. (1999), a reduction of 3.3% in the frequency of proline-allele carriers among centenarians was reported. This reduction is consistent with their hypothesis, although the authors suggest that it is not (Bonafe et al. 1999). Moreover, our corresponding projected reduction in allelic frequency is 2.8% for a relative risk of 1.5; Bonafe et al. (1999) observed a 2.7% reduction in frequency. However, to have, at a significance level of .05, 80% power to detect a 3.0%–4.8% reduction in the prevalence of proline-allele carriers, a minimum of 2,002–3,178 people would be needed, given a relative risk of 2.0; and 5,176–8,182 would be needed, given a relative risk of 1.5 (results of power calculations are given in table 2). n n n nTable 1 n nSimulated Relative-Risk Calculation for a Standard Population of 1,000 Persons[Note] n n n n n nTable 2 n nPower Calculations n n n nNotwithstanding these conclusions and observations, we believe that p53 allelism is an important cancer-susceptibility factor but that it is more complex than that simply defined by the polymorphism at codon 72. A series of studies by a Swedish group (Beckman et al. 1994; Birgander et al. 1995, 1996a, 1996b; Sjalander et al. 1995a, 1995b, 1996a, 1996b) indicated that a constellation of three p53 polymorphisms (intron 3, codon 72, and intron 6) constituted a haplotype predictive of increased breast cancer risk (odds ratio [OR] = 2.9, 95% confidence interval [CI] = 1.4–6.3 [Sjalander et al. 1996a]). Haplotypes were deduced on the basis of population frequencies of the individual polymorphisms. First, estimates of pairwise haplotype frequencies were calculated, and extended haplotype frequencies were deduced from this information. A subsequent study examined the same question but used a PCR-based method for physical determination of the haplotypes (Weston et al. 1997, 1998). The results of the latter study were consistent with those reported in the study by Sjalander et al. (1996a) (OR = 2.5, 95% CI = 1.3–4.8, in postmenopausal women) and further implicated a specific haplotype, designated “p531-2-1.” More recently, a third study, estimating extended haplotypes of these three polymorphisms in a German population, provided results consistent with an elevated breast cancer risk associated with inheritance of p531-2-1 (OR = 2.0, 95% CI = 1.0–3.9 [Wang-Gohrke et al. 1998]). It is to be noted that this latest report has adopted an allelic nomenclature different from that developed by Beckman et al. (1994) and adopted by others (Sjalander et al. 1996a; Weston et al. 1997, 1998). n nThe balance of the results of human breast cancer studies of molecular epidemiological haplotype association—specifically, three positive associations in three studies—suggests that inheritance of the p531-2-1 haplotype does have an impact on human breast cancer risk (Sjalander et al. 1996a; Wang-Gohrke et al. 1998; Weston et al. 1998). However, more research is needed to resolve this question. In our laboratory, we have adopted a complementary molecular epidemiological and basic-science approach. First, we are trying again to replicate the epidemiological studies of breast cancer, by using the PCR-based physical haplotype method in a population-based case-control study that has sufficient power. Second, we have derived normal human mammary cell strains with known haplotypes and plan to test their response to suspect breast carcinogens.

Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene‐DNA (BP‐DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 μM BP for 24 hr in the presence or absence of 5 μM CHLN, we measured BP‐DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP‐induced up‐regulation of CYP1A1 and CYP1B1 expression, as well as up‐regulation of groups of interferon‐inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT‐PCR) the maximal inhibition of BP‐induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin‐O‐deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN‐exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP‐exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced. Environ. Mol. Mutagen. 2009. Published 2009 Wiley‐Liss, Inc.

Application of Oligonucleotide Microarray Technology to Toxic Occupational Exposures

Microarray technology has advanced toward analysis of toxic occupational exposures in biological systems. Microarray analysis is an ideal way to search for biomarkers of exposure, even if no specific gene or pathway has been identified. Analysis may now be performed on thousands of genes simultaneously, as opposed to small numbers of genes as in the past. This ability has been put to use to analyze gene expression profiles of a variety of occupational toxins in animal models to classify toxins into specific categories based on response. Analysis of normal human cell strains allows an extension of this analysis to investigate the role of interindividual variation in response to various toxins. This methodology was used to analyze four occupationally related toxins in our lab: oxythioquinox (OTQ), a quinoxaline pesticide; malathion, an organophosphate pesticide; di-n-butyl phthalate (DBP), a chemical commonly found in personal care and cosmetic items; and benzo[a]pyrene (BaP), an environmental and occupational carcinogen. The results for each exposure highlighted signaling pathways involved in response to these occupational exposures. Both pesticides showed increase in metabolic enzymes, while DBP showed alterations in genes related to fertility. BaP exposure showed alterations in two cytochrome P450s related to carcinogenicity. When used with occupational exposure information, these data may be used to augment risk assessment to make the workplace safer for a greater proportion of the workforce, including individuals susceptible to disease related to exposures.

Chronic Beryllium Disease, HLA-DPB1, and the DP Peptide Binding Groove

Multiple epidemiologic studies demonstrate associations between chronic beryllium disease (CBD), beryllium sensitization (BeS), and HLA-DPB1 alleles with a glutamic acid residue at position 69 (E69). Results suggest that the less-frequent E69 variants (non-*0201/*0202 alleles) might be associated with greater risk of CBD. In this study, we sought to define specific E69-carrying alleles and their amino acid sequences in the DP peptide binding groove, as well as their relationship to CBD and BeS risk, using the largest case control study to date. We enrolled 502 BeS/CBD subjects and 653 beryllium-exposed controls from three beryllium industries who gave informed consent for participation. Non-Hispanic white cases and controls were frequency-matched by industry. HLA-DPB1 genotypes were determined using sequence-specific primer PCR. The E69 alleles were tested for association with disease individually and grouped by amino acid structure using logistic regression. The results show that CBD cases were more likely than controls to carry a non-*02 E69 allele than an *02 E69, with odds ratios (95% confidence interval) ranging from 3.1 (2.1–4.5) to 3.9 (2.6–5.9) (p < 0.0001). Polymorphic amino acids at positions 84 and 11 were associated with CBD: DD versus GG, 2.8 (1.8–4.6), p < 0.0001; GD versus GG, 2.1 (1.5–2.8), p < 0.0001; LL versus GG, 3.2 (1.8–5.6), p < 0.0001; GL versus GG, 2.8 (2.1–3.8), p < 0.0001. Similar results were found within the BeS group and CBD/BeS combined group. We conclude that the less frequent E69 alleles confer more risk for CBD than does *0201. Recent studies examining how the composition and structure of the binding pockets influence peptide binding in MHC genes, as well of studies showing the topology of the TCR to likely bind DPB1 preferentially, give plausible biological rationale for these findings.

Genetic determinants of sensitivity to beryllium in mice

Chronic beryllium disease (CBD), an irreversible, debilitating granulomatous lung disease is caused by exposure to beryllium. This occupational hazard occurs in primary production and machining of Be-metal, BeO, beryllium - containing alloys, and other beryllium products. CBD begins as an MHC Class II-restricted, TH1 hypersensitivity, and the Human Leukocyte Antigen, HLA-DPB1E69, is associated with risk of developing CBD. Because inbred strains of mice have not provided good models of CBD to date, three strains of HLA-DPB1 transgenic mice in an FVB/N background were developed; each contains a single allele of HLA-DPB1 that confers a different magnitude of risk for chronic beryllium disease: HLA-DPB1*0401 (OR ≈ 0.2), HLA-DPB1*0201 (OR ≈ 3), and HLA-DPB1*1701 (OR ≈ 46). The mouse ear swelling test (MEST) was employed to determine if these different alleles would support a hypersensitivity response to beryllium. Mice were first sensitized on the back and subsequently challenged on the ear. In separate experiments, mice were placed into one of three groups (sensitization/challenge): C/C, C/Be, and Be/Be. In the HLA-DPB1*1701 mice, the strain with the highest risk transgene, the Be/Be group was the only group that displayed significant maximum increased ear thickness of 19.6% ± 3.0% over the baseline measurement (pu2009<u20090.05). No significant changes were observed in the other transgenic strains for any treatment condition. In addition, inter-strain differences in response to beryllium in seven inbred strains were investigated through use of the MEST, these included: FVB/N, AKR, Balb/c, C3H/HeJ, C57/BL6, DBA/2, and SJL/J. The FVB/N strain was least responsive, while the SJL/J and C57/BL6 strains were the highest responders. Our results suggest that the HLA-DPB1*1701 transgene product is an important risk factor for induction of the beryllium-sensitive phenotype. This model should be a useful tool for investigating beryllium sensitization.

Polycyclic Aromatic Hydrocarbon Biomarkers of Internal Exposure in U.S. Army Soldiers Serving in Kuwait in 1991

Abstract Biomarkers of exposure were applied to a cohort of U.S. Army soldiers who were deployed to Kuwait and Saudi Arabia in 1991 in the aftermath of the Persian Gulf War. The U.S. Army Environmental Hygiene Agency (currently the U.S Army Center for Health Promotion and Preventive Medicine) monitored air and soil for ambient PAHs. In addition, a group of 61 soldiers kept diaries of daily activities. These soldiers provided blood and urine samples in June in Germany before deployment to Kuwait, in August after 8 weeks in Kuwait, and in October, one month after the return to Germany. DNA, prepared from white blood cells, was assayed for PAH-DNA adducts by immunoassay and bulky aromatic adducts by 32P-postlabeling. Urinary 1-hydroxypyrene-glucuronide (1-OH-PG) was determined by synchronous fluorescence spectrometry. Contrary to expectations, environmental monitoring showed low ambient PAH levels in the areas where these soldiers were working in Kuwait. In addition, literature values for ambient PAH monitor...

Correlation between CYP1A1 transcript, protein level, enzyme activity and DNA adduct formation in normal human mammary epithelial cell strains exposed to benzo[a]pyrene

UNLABELLEDnThe polycyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BP) is thought to bind covalently to DNA, through metabolism by cytochrome P450 1A1 (CYP1A1) and CYP1B1, and other enzymes, to form r7, t8, t9-trihydroxy-c-10-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]-pyrene (BPdG). Evaluation of RNA expression data, to understand the contribution of different metabolic enzymes to BPdG formation, is typically presented as fold-change observed upon BP exposure, leaving the actual number of RNA transcripts unknown. Here, we have quantified RNA copies/ng cDNA (RNA cpn) for CYP1A1 and CYP1B1, as well asnnnNAD(P)Hnquinone oxidoreductase 1 (NQO1), which may reduce formation of BPdG adducts, using primary normal human mammary epithelial cell (NHMEC) strains, and the MCF-7 breast cancer cell line. In unexposed NHMECs, basal RNA cpn values were 58-836 for CYP1A1, 336-5587 for CYP1B1 and 5943-40112 for NQO1. In cells exposed to 4.0 µM BP for 12h, RNA cpn values were 251-13234 for CYP1A1, 4133-57078 for CYP1B1 and 4456-55887 for NQO1. There were 3.5 (mean, range 0.2-15.8) BPdG adducts/10(8) nucleotides in the NHMECs (n = 16), and 790 in the MCF-7s. In the NHMECs, BP-induced CYP1A1 RNA cpn was highly associated with BPdG (P = 0.002), but CYP1B1 and NQO1 were not. Western blots of four NHMEC strains, chosen for different levels of BPdG adducts, showed a linear correlation between BPdG and CYP1A1, but not CYP1B1 or NQO1. Ethoxyresorufin-O-deethylase (EROD) activity, which measures CYP1A1 and CYP1B1 together, correlated with BPdG, but NQO1 activity did not. Despite more numerous levels of CYP1B1 and NQO1 RNA cpn in unexposed and BP-exposed NHMECs and MCF-7cells, BPdG formation was only correlated with induction of CYP1A1 RNA cpn. The higher level of BPdG in MCF-7 cells, compared to NHMECs, may have been due to a much increased induction of CYP1A1 and EROD. Overall, BPdG correlation was observed with CYP1A1 protein and CYP1A1/1B1 enzyme activity, but not with CYP1B1 or NQO1 protein, or NQO1 enzyme activity.

Human inter-individual variability in metabolism and genotoxic response to zidovudine

A mainstay of the antiretroviral drugs used for therapy of HIV-1, zidovudine (AZT) is genotoxic and becomes incorporated into DNA. Here we explored host inter-individual variability in AZT-DNA incorporation, by AZT radioimmunoassay (RIA), using 19 different strains of normal human mammary epithelial cells (NHMECs) exposed for 24 h to 200 microM AZT. Twelve of the 19 NHMEC strains showed detectable AZT-DNA incorporation levels (16 to 259 molecules of AZT/10(6) nucleotides), while 7 NHMEC strains did not show detectable AZT-DNA incorporation. In order to explore the basis for this variability, we compared the 2 NHMEC strains that showed the highest levels of AZT-DNA incorporation (H1 and H2) with 2 strains showing no detectable AZT-DNA incorporation (L1 and L2). All 4 strains had similar (> or =80%) cell survival, low levels of accumulation of cells in S-phase, and no relevant differences in response to the direct-acting mutagen bleomycin (BLM). Finally, when levels of thymidine kinase 1 (TK1), the first enzyme in the pathway for incorporation of AZT into DNA, were determined by Western blot analysis in all 19 NHMEC strains at 24 h of AZT exposure, higher TK1 protein levels were found in the 12 strains showing AZT-DNA incorporation, compared to the 7 showing no incorporation (p=0.0005, Mann-Whitney test). Furthermore, strains L1 and L2, which did not show AZT-DNA incorporation at 24 h, did have measurable incorporation by 48 and 72 h. These data suggest that variability in AZT-DNA incorporation may be modulated by inter-individual differences in the rate of induction of TK1 in response to AZT exposure.