Mary A. Watson

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5′-phosphate and 3′-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1−/− mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β (pol β) is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1−/−, and to a lesser extent in pol β−/− cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol β−/− cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1−/− cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

p53 mutation hotspot in radon-associated lung cancer

Mutations in gene p53 are the most common defects in lung cancer and may be a pathway through which environmental carcinogens initiate cancer. We investigated p53 mutations in lung cancers from uranium miners with high radon exposure. 16 (31%) of 52 large-cell and squamous-cell cancers from miners contained the same AGG to ATG transversion at codon 249, including cancers from 3 or 5 miners who had never smoked. This specific mutation has been reported in only 1 of 241 published p53 mutations from lung cancers. The codon 249 mutation may be a marker for radon-induced lung cancer.

XRCC1 polymorphisms and head and neck cancer.

Inter-individual differences in DNA repair capacity have been demonstrated using a variety of phenotypic assays, including reduced repair among patients with squamous cell carcinoma of the head and neck (SCCHN). The XRCC1 DNA repair gene may facilitate DNA strand break and base excision repair. A recent case-control study of SCCHN reported associations with two polymorphisms of the XRCC1 including the exon 6, 194Arg/Arg genotype and the exon 10, 399 Gln/Gln genotype. We conducted an analysis of these two XRCC1 polymorphisms using data from a case-control study of SCCHN. Among white subjects, we found a weak elevation in risk associated with the Arg194Trp polymorphism [odds ratio (OR)=1.3; 95% confidence interval (CI)=0.6-2.9] and a decreased risk for the Arg399Gln polymorphism (OR=0.6; CI=0.4-1.1). We found a markedly decreased odds ratio for the Gln/Gln genotype among whites (OR=0.1; CI=0.04-0.6) and blacks (OR=0.01; CI=0.0004-0.3). We also found a suggestion of an interaction between the Arg194Trp and Arg399Gln polymorphisms and tobacco use. Additional epidemiologic and functional studies are needed to resolve the importance of these XRCC1 polymorphisms in SCCHN.

Association Between Blood Lead and the Risk of Amyotrophic Lateral Sclerosis

The authors conducted a 2003-2007 case-control study including 184 cases and 194 controls to examine the association between blood lead and the risk of amyotrophic lateral sclerosis (ALS) among US veterans and to explore the influence on this association of bone turnover and genetic factors related to lead toxicokinetics. Blood lead, plasma biomarkers of bone formation (procollagen type 1 amino-terminal peptide (PINP)) and resorption (C-terminal telopeptides of type 1 collagen (CTX)), and the K59N polymorphism in the delta-aminolevulinic acid dehydratase gene, ALAD, were measured. Odds ratios and 95% confidence intervals for the association of blood lead with ALS were estimated with unconditional logistic regression after adjustment for age and bone turnover. Blood lead levels were higher among cases compared with controls (P < 0.0001, age adjusted). A doubling of blood lead was associated with a 1.9-fold increased risk of ALS (95% confidence interval: 1.3, 2.7) after adjustment for age and CTX. Additional adjustment for PINP did not alter the results. Significant lead-ALS associations were observed in substrata of PINP and CTX levels. The K59N polymorphism in the ALAD gene did not modify the lead-ALS association (P = 0.32). These results extend earlier findings by accounting for bone turnover in confirming the association between elevated blood lead level and higher risk of ALS.

Carotenoids/vitamin C and smoking-related bladder cancer

Previous epidemiological studies of fruit and vegetable intake and bladder cancer risk have yielded inconsistent results, especially with respect to the role of cigarette smoking as a possible modifier of the diet‐bladder cancer association. A population‐based case‐control study was conducted in nonAsians of Los Angeles, California, which included 1,592 bladder cancer patients and an equal number of neighborhood controls matched to the index cases by sex, date of birth (within 5 years) and race between January 1, 1987 and April 30, 1996. Information on smoking, medical and medication history, and intake frequencies of food groups rich in preformed nitrosamines, vitamins A and C and various carotenoids, were collected through in‐person, structured interviews. Beginning in January 1992, all case patients and their matched control subjects were asked for a blood sample donation at the end of the in‐person interviews for measurements of 3‐ and 4‐aminobiphenyl (ABP) hemoglobin adducts, and glutathione S‐transferases M1/T1/P1 (GSTM1/T1/P1) and N‐acetyltransferase‐1 (NAT1) genotypes. Seven hundred seventy‐one (74%) case patients and 775 (79%) control subjects consented to the blood donation requests. In addition, all case patients and matched control subjects were asked to donate an overnight urine specimen following caffeine consumption for measurements of cytochrome P4501A2 (CYP1A2) and N‐acetyltransferase‐2 (NAT2) phenotypes. Urine specimens were collected from 724 (69%) case patients and 689 (70%) control subjects. After adjustment for nondietary risk factors including cigarette smoking, there were strong inverse associations between bladder cancer risk and intake of dark‐green vegetables [p value for linear trend (p) = 0.01], yellow‐orange vegetables (p = 0.01), citrus fruits/juices (p = 0.002) and tomato products (p = 0.03). In terms of nutrients, bladder cancer risk was inversely associated with intake of both total carotenoids (p = 0.004) and vitamin C (p = 0.02). There was a close correlation (r = 0.58, p = 0.0001) between intakes of total carotenoids and vitamin C in study subjects. When both nutrients were included in a multivariate logistic regression model, only total carotenoids exhibited a residual effect that was of borderline statistical significance (p = 0.07 and p = 0.40 for total carotenoids and vitamin C, respectively). Cigarette smoking was a strong modifier of the observed dietary effects; these protective effects were confined largely to ever smokers and were stronger in current than ex‐smokers. Smokers showed a statistically significant or borderline statistically significant decrease in 3‐ and 4‐aminobiphenyl (ABP)‐hemoglobin adduct level with increasing intake of carotenoids (p = 0.04 and 0.05, respectively). The protective effect of carotenoids on bladder cancer seemed to be influenced by NAT1 genotype, NAT2 phenotype and CYP1A2 phenotype; the association was mainly confined to subjects possessing the putative NAT1‐rapid, NAT2‐rapid and CYP1A2‐rapid genotype/phenotype. The carotenoid‐bladder cancer association was not affected by the GSTM1, GSTT1 and GSTP1 genotypes.

Genetic determinants in the metabolism of bladder carcinogens in relation to risk of bladder cancer

Genetically determined factors that alter the metabolism of tobacco carcinogens can influence an individuals susceptibility to bladder cancer. The associations between the genotypes of glutathione S-transferase (GST) M1, GSTP1, GSTT1 and N-acetyltransferase (NAT) 1 and the phenotypes of NAT2 and cytochrome P450 (CYP) 1A2 and bladder cancer risk were examined in a case-control study involving 731 bladder cancer patients and 740 control subjects in Los Angeles County, California. Individual null/low-activity genotypes of GSTM1, GSTT1 and GSTP1 were associated with a 19-48% increase in odds ratio (OR) of bladder cancer. The strongest association was noted for GSTM1 [OR for the null genotype = 1.48, 95% confidence interval (CI) = 1.19-1.83]. When the three GST genes were examined together, there was a monotonic, statistically significant association between increasing number of null/low-activity genotypes and risk (P for trend = 0.002). OR (95% CI) for one and two or more null/low-activity GST genotypes was 1.42 (1.12-1.81) and 1.71 (1.25-2.34), respectively, relative to the absence of null/low-activity GST genotype. NAT2 slow acetylation was associated with doubled risk of bladder cancer among individuals with known high exposures to carcinogenic arylamines (OR = 2.03, 95% CI = 1.12-3.69, P = 0.02). The effect of NAT2 slow acetylation was even stronger in the presence of two or more null/low-activity GST genotypes. There were no associations between bladder cancer risk and NAT1 genotype or CYP1A2 phenotype.

A pilot study investigating the role of NAT1 and NAT2 polymorphisms in gastric adenocarcinoma

In humans, aromatic and heterocyclic amine carcinogens may be acetylated by the expression products of either of the N‐acetyltransferase genes, NAT1 or NAT2. This conjugation reaction can result in either activation or detoxication of these carcinogens depending on the tissue involved. Recent studies suggest that polymorphisms in NAT1 or NAT2 may modulate cancer risk. To determine if genetic differences in NAT1 and NAT2 could alter risk of gastric cancer, we tested for the presence of polymorphic N‐acetyltransferase alleles (both NAT1 and NAT2) in a preliminary study of 94 gastric adenocarcinoma patients and 112 control subjects from North Staffordshire, England. We used established PCR protocols to genotype for NAT2 and NAT1 alleles (NAT2*4, NAT2*5, NAT2*6, NAT2*7, NAT2*14; NAT1*3, NAT1* 4, NAT1*10, and NAT1*11), and implemented an oligonucleotide ligation assay (OLA) to test for low‐activity NAT1 alleles [NAT1*14 (G560A), NAT1*15 (C559T), and NAT1*17 (C190T)]. No significant increased risk was observed for NAT2 acetylation genotypes. However, among all cases, we found that individuals inheriting a variant NAT1 allele, NAT1*10, have a significantly elevated risk for gastric cancer (OR = 2.2, 95% CI 1.2–3.9, P < 0.01). Interestingly, the risk observed for NAT1*10 appears to be solely associated with advanced‐stage tumors (OR = 4.8, P < 0.001), suggesting a possible role in progression to advanced disease. This preliminary finding needs confirmation in a larger, detailed epidemiological study. Int. J. Cancer 87:507–511, 2000.

Pilot study of free and conjugated urinary mutagenicity during consumption of pan-fried meats: possible modulation by cruciferous vegetables, glutathione S-transferase-M1, and N-acetyltransferase-2

Epidemiological and experimental evidence indicates that consumption of fried meats in conjunction with certain genotypes of phase I and II metabolism genes poses an elevated risk for colorectal cancer. Parallel to this, the consumption of cruciferous vegetables is associated with a reduced risk of colon cancer. Therefore, we designed a 6-week pilot feeding study to evaluate the effect of these variables on urinary mutagenicity, which is a biomarker associated with fried-meat consumption. Eight subjects were fed fried meats daily for six weeks; four ate cruciferous vegetables, and four ate non-cruciferous vegetables. Urinary mutagenicity was evaluated in the presence of S9 in strain YG1024 of Salmonella, which is a frameshift strain that overproduces acetyltransferase. C18/methanol extracts of 24-h urines collected once each week were tested unhydrolyzed (free mutagenicity) and hydrolyzed (total mutagenicity); the difference between the two was the conjugated mutagenicity. Although not significant, the levels of conjugated urinary mutagenicity doubled among crucifera consumers and decreased to 30% of the initial levels among non-crucifera consumers, suggesting the possibility that crucifera may enhance the level of conjugated urinary mutagenicity resulting from consumption of fried meats. Such an effect would be consistent with the documented ability of cruciferous vegetables to induce phase II enzymes. The NAT2 rapid phenotype was significantly associated with approximately 2-fold increases in conjugated (p = 0.05) and total (p = 0.004) urinary mutagenicity relative to NAT2 slow subjects, consistent with the elevated risk confirmed by the NAT2 rapid phenotype for colorectal cancer among meat consumers. An approximately 2-fold increase in urinary mutagenicity among the GSTM1- subjects relative to the GSTM1+ subjects approached significance for free (p = 0.18) and total (p = 0.13) urinary mutagenicity. This is the first report on (a) the mutagenicity of hydrolyzed urine, which was consistently more mutagenic than unhydrolyzed urine; (b) the potential enhancement of conjugated urinary mutagenicity by crucifera; and (c) the association of the rapid NAT2 and possibly the GSTM1- phenotype with elevated levels of fried meat-associated urinary mutagenicity.

Bayesian Latent Variable Models for Median Regression on Multiple Outcomes

Often a response of interest cannot be measured directly and it is necessary to rely on multiple surrogates, which can be assumed to be conditionally independent given the latent response and observed covariates. Latent response models typically assume that residual densities are Gaussian. This article proposes a Bayesian median regression modeling approach, which avoids parametric assumptions about residual densities by relying on an approximation based on quantiles. To accommodate within-subject dependency, the quantile response categories of the surrogate outcomes are related to underlying normal variables, which depend on a latent normal response. This underlying Gaussian covariance structure simplifies interpretation and model fitting, without restricting the marginal densities of the surrogate outcomes. A Markov chain Monte Carlo algorithm is proposed for posterior computation, and the methods are applied to single-cell electrophoresis (comet assay) data from a genetic toxicology study.

Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells.

The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE‐B3) were severely damaged when incubated with berberine (25 μM) and exposed to UVA (5 J cm−2). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N‐acetylcysteine (5 mM). When exposed to UVA (5 J cm−2) both berberine (10 μM) and palmatine (10 μM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (λ > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal are estimated to contain ∼1 mM berberine, while in tinctures the alkaloid concentration may be more than 10 times higher. Our findings show that eyewashes and lotions derived from Goldenseal or containing berberine must be used with caution when the eyes are exposed to bright sunlight but that oral preparations are not likely to cause ocular phototoxicity.