Simonetta Gemma

Polymorphic DNA repair and metabolic genes: a multigenic study on gastric cancer

Risk factors for gastric cancer (GC) include inter-individual variability in the inflammatory response to Helicobacter pylori infection, in the ability of detoxifying DNA reactive species and repairing DNA damage generated by oxidative stress and dietary carcinogens. To evaluate the association between polymorphic DNA repair genes and GC risk, a case-control study including 314 histologically confirmed GC patients and 548 healthy controls was conducted in a GC high-risk area in Tuscany, Italy. Polymorphic variants of base excision repair (APE1-D148E, XRCC1-R194W, XRCC1-R399Q and OGG1-S326C), nucleotide excision repair (XPC-PAT, XPA-23G>A, ERCC1-19007T>C and XPD-L751Q), recombination (XRCC3-T241M) and alkylation damage reversal (MGMT-L84F) were tested for their potential role in the development of GC by using logistic regression models. The same population was also characterised for GSTT1 and GSTM1 variant alleles to search for possible functional interactions between metabolic and DNA repair genotypes by two-way interactions using multivariate logistic models. No significant association between any single DNA repair genotype and GC risk was detected with a borderline association with the XPC-PAT homozygous genotype [odds ratio (OR) =1.42; 95% confidence interval (CI) 0.94-2.17]. Gene-gene interaction analysis revealed combinations of unfavourable genotypes involving either multiple DNA repair polymorphisms or DNA repair and GST-specific genotypes. The combination of the XPC-PAT and the XPA variant alleles significantly increased GC risk (OR=2.15; 95% CI 1.17-3.93, P=0.0092). A significant interaction was also found between the APE1 wild-type genotype and either the single GSTT1 (OR=4.90; 95% CI 2.38-10.11, P=0.0079) or double GSTM1-GSTT1 null (OR=7.84; 95% CI 3.19-19.22, P=0.0169) genotypes or the XPA-mutant allele (OR=3.56; 95% CI 1.53-8.25, P=0.0012). These findings indicate that a complex interaction between host factors such as oxidative stress, antioxidant capacity and efficiency of multiple DNA repair pathways underlies the inter-individual variability in GC risk.

Interleukin-1 gene polymorphisms and gastric cancer risk in a high-risk Italian population

OBJECTIVES:Host genetic factors, including the IL1 gene cluster, play a key role in determining the long-term outcome of Helicobacter pylori infection. The aim of the study was to investigate the relationship between selected IL1 loci polymorphisms and gastric cancer risk in an Italian population.METHODS:In a case-control study we compared the IL1B−31 and IL1B+3954 biallelic and IL1RN pentaallelic variable number of tandem repeats (VNTR) polymorphisms in 185 gastric cancer patients and 546 controls randomly sampled from the general population of an area at high gastric cancer risk (Tuscany, Central Italy).RESULTS:Genotype frequencies of the IL1B−31 T/C, IL1B+3954 C/T, and IL1RN polymorphisms among our population controls were in Hardy-Weinberg equilibrium. In multivariate analyses, no increase in gastric cancer risk was observed for the IL1B−31*C− and IL1B+3954*T− carriers; a significant 50% increase emerged for IL1RN*2 allele carriers (OR = 1.49; 95% CI: 1.01–2.21).Analyses based on combined genotypes showed also that the association with IL1RN*2 allele was limited to two-variant allele carriers who were also homozygous for the IL1B−31*T allele (OR = 2.23; 95% CI: 1.18–4.23) with a statistically significant interaction between these two genotypes (p= 0.043). Haplotype analysis showed an increased risk for the haplotype IL1RN*2/IL1B−31*T.CONCLUSIONS:Our results suggest that host genetic factors (such as the IL1RN and the IL1B−31 polymorphisms) interact in the complex process of gastric carcinogenesis in this high-risk Italian population. Overall, this effect appears more modest than previously reported in other populations, supporting the hypothesis that other still-to-be-defined factors are important in gastric carcinogenesis. These findings might be due to a haplotype effect.

GSTT1 and GSTM1 gene polymorphisms and gastric cancer in a high-risk italian population

Glutathione S‐Transferases (GSTs) are a family of phase II enzymes involved in the detoxification of potential carcinogens and provided of a strong antioxidant function by neutralizing electrophiles and free radicals. The GSTM1 and GSTT1 isoenzymes exhibit deletion polymorphisms, resulting in a lack of activity, and the null genotypes have been associated with increased cancer risk at several sites, including the stomach, although with contrasting results. We carried out a case‐control study to evaluate whether these polymorphisms modulate the risk of developing gastric cancer (GC). Genotypes for GSTM1 and GSTT1 were obtained from a series of 175 histologically confirmed GC patients and a large series of 546 healthy controls randomly sampled from the general population of Tuscany, an area at high GC risk. No difference in the frequency of GSTM1 null genotype was observed between cases and controls, whereas the GSTT1 null genotype was more frequent among cases (p = 0.04). Multivariate single‐gene analyses adjusted for possible confounders showed that the GSTT1 null genotype, but not the GSTM1 null genotype, was associated with an increased GC risk. Combined‐genotype analyses showed a significantly increased GC risk only for the double null (GSTM1‐GSTT1) genotype (OR = 2.27; 95% CI: 1.14–4.53). A statistically significant positive interaction between the 2 null genotypes was observed (p = 0.02). Our findings suggest that only subjects lacking both GSTM1 and GSTT1 activity are at increased GC risk. This study provides further support to the hypothesis that the risk of developing GC is influenced by inter‐individual variation in both carcinogen detoxification and antioxidant capacity.

Metabolic and genetic factors contributing to alcohol induced effects and fetal alcohol syndrome

Alcohol-related damages on newborns and infants include a wide variety of complications from facial anomalies to neurodevelopmental delay, known as fetal alcohol syndrome (FAS). However, only less than 10% of women drinking alcohol during pregnancy have children with FAS. Understanding the risk factors increasing the probability for newborn exposed in utero to alcohol to develop FAS is therefore a key issue. The involvement of genetics as a one risk factor in FAS has been suggested by animal models and by molecular epidemiological studies on different populations, bearing allelic variants for those enzymes, such as ADH e CYP2E1, involved in ethanol metabolism. Indeed, one of the major factors determining the peak blood alcohol exposure to the fetus is the metabolic activity of the mother, in addition to placental and fetal metabolism, explaining, at least partially, the risk of FAS. The different rates of ethanol metabolism may be the result of genetic polymorphisms, the most relevant of which have been described in the paper.

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver.

The metabolism of diazinon, an organophosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague–Dawley (SD) rats or SD rats treated with different P450‐specific inducers (phenobarbital, dexamethasone, β‐napthoflavone, and pyrazole).

Health risk evaluation associated to Planktothrix rubescens: An integrated approach to design tailored monitoring programs for human exposure to cyanotoxins

Increasing concern for human health related to cyanotoxin exposure imposes the identification of pattern and level of exposure; however, current monitoring programs, based on cyanobacteria cell counts, could be inadequate. An integrated approach has been applied to a small lake in Italy, affected by Planktothrix rubescens blooms, to provide a scientific basis for appropriate monitoring program design. The cyanobacterium dynamic, the lake physicochemical and trophic status, expressed as nutrients concentration and recycling rates due to bacterial activity, the identification/quantification of toxic genotype and cyanotoxin concentration have been studied. Our results indicate that low levels of nutrients are not a marker for low risk of P. rubescens proliferation and confirm that cyanobacterial density solely is not a reliable parameter to assess human exposure. The ratio between toxic/non-toxic cells, and toxin concentrations, which can be better explained by toxic population dynamic, are much more diagnostic, although varying with time and environmental conditions. The toxic fraction within P. rubescens population is generally high (30-100%) and increases with water depth. The ratio toxic/non-toxic cells is lowest during the bloom, suggesting a competitive advantage for non-toxic cells. Therefore, when P. rubescens is the dominant species, it is important to analyze samples below the thermocline, and quantitatively estimate toxic genotype abundance. In addition, the identification of cyanotoxin content and congeners profile, with different toxic potential, are crucial for risk assessment.

Bioactivation of chloroform in hepatic microsomes from rodent strains susceptible or resistant to CHCl3 carcinogenicity.

The dependence of adduct formation on oxygen concentration and glutathione (GSH) presence was used to characterize the bioactivation of chloroform in hepatic microsomes of Sprague-Dawley and Osborne-Mendel rats and B6C3F1 and C57Bl/6J mice. Both oxidative and reductive pathways were present in all the animals tested. Oxidative activation, very sensitive to oxygen withdrawal, was the major pathway responsible for the covalent binding to microsomal proteins and lipids at 0.1 mM CHCl3. The relative contribution of either pathway to the covalent binding to microsomal lipids at 5 mM CHCl3 was dependent on the oxygen concentration. At 1% pO2, i.e., in the range of the hepatic physiological oxygenation level, B6C3F1 mouse hepatic microsomes showed an oxidative activation distinctly higher than that of hepatic microsomes of other rodents; on the other hand, reductive activation was present only in B6C3F1 mouse and Osborne-Mendel rat liver microsomes. The reductive intermediates were the only contributors to the covalent binding of CHCl3 equivalents to lipids in the presence of GSH; indeed the reactive intermediates produced by the oxidative pathway were fully scavenged by this compound. These results are discussed with respect to the species specificity of CHCl3 hepatocarcinogenesis.

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism

The two CHCl3 activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and beta-naphthoflavone). The present results provide direct evidence that CHCl3 concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl3 concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10-15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl3 only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl3 oxidative bioactivation do not participate in CHCl3 reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl3 activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity.

Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice

Abstract The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl3. Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl3 oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl3 bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.

In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains.

We have comparatively studied in vitro the oxidative and reductive pathways of chloroform metabolism in hepatic and renal microsomes of rodent strains used for carcinogenicity testing (B6C3F1 mice, Osborne Mendel and Sprague Dawley rats). To this aim we exploited the regioselective binding of phosgene to phospholipid (PL) polar heads and of dichloromethyl radical to PL fatty acyl chains, using a method based on the chemical transmethylation of PL adducts, followed by phase partitioning of the resulting products (De Biasi et al., 1992). The analysis of results let us to conclude at first that a (14)C label partitioning by 89.2 (±6.5)% or 13.7 (±5.0)% in the aqueous phase is typical of the PL adduct with phosgene (PL-PHOS) or with dichloromethyl radical (PL-RAD), respectively. Metabolism of 0.1 mM CHCl(3) was mainly oxidative in all the samples, being hepatic microsomes more active than renal ones by about one order of magnitude and levels of CHCl(3)-derived PL adducts in B6C3F1 mouse liver microsomes higher than in rat samples. At 5 mM CHCl(3), total levels of PL adducts in renal microsomes reached levels almost similar to those found in liver microsomes. However, while B6C3F1 mouse kidney microsomes produced both reactive metabolites, similarly as the hepatic samples, Osborne Mendel rat kidney microsomes bioactivated CHCl(3) only reductiveiy, producing the radical. The relevance of this finding depends on the fact that phosgene is known to be the major cause of CHCl(3) toxicity, based on data with the rat liver and mouse liver and kidney, while nephrotoxicity in rats occurs with minimal production of COCl(2). Chloroform reductive bioactivation may therefore provide a reasonable explanation for the toxicity of chloroform to the rat kidney. The same finding may be of interest in elucidating the metabolic reasons of the chloroform-induced kidney tumors in Osborne Mendel rats.