Subacute intoxication with sodium nitrate induces hematological and biochemical alterations and liver injury in male Wistar rats.

Nitrate pollution has emerged as a problem of great importance because in recent years, the levels of nitrate in soil and groundwater have increased, mainly through anthropogenic activities, such as the use of fertilizers in agriculture, domestic wastewater and septic tanks, industrial waste and deforestation. In animals, nitrate reduction to nitrite (NO2) and nitric oxide (NO) promote the formation of methemoglobin in the blood and the generation of highly reactive intermediates that induce oxidative stress in target organs. Exposition to nitrates has been associated with methemoglobinemia, reproductive toxicity, metabolic and endocrine alterations and cancer. This study analyzed acute intoxication with sodium nitrate (NaNO3) in male Wistar rats, aged 12-16 weeks. Four groups with n = 10 rats each were formed: group 1 was the control, and group 2, group 3 and group 4 were treated for 10 days with intragastric doses of 19, 66 and 150 mg/kg/d NaNO3, respectively. Hematological, metabolic and histological biomarkers in the liver were analyzed. The results showed high percentages of methemoglobin, an increase in NO2 in the plasma and an accumulation in the liver. Moreover, there were high counts of white blood cells and platelets in all treated groups. Additionally, there was an increase in the spleen weight in group 4. High levels of glucose, triglycerides, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were observed and were significantly increased in groups 3 and 4. For oxidative stress biomarkers, there were increases in Thiobarbituric Acid Reactive Substances (TBARS), total GSH and SOD activity, mainly in group 4. Changes in mitochondrial activity were not significant. Histopathological analyses of the liver showed inflammation, infiltration of mononuclear cells, steatosis, ischemia and necrosis, and these findings were more evident at high doses of NaNO3 in which high of S-nitrosylation were found. In conclusion, NaNO3 was reduced to NO2, thereby inducing methemoglobinemia, whereas other reactive species generated oxidative stress, causing hematological and metabolic alterations and injury to the liver.

Differences in 4-hydroxyestradiol levels in leukocytes are related to CYP1A1(∗)2C, CYP1B1(∗)3 and COMT Val158Met allelic variants.

Exposure to estrogen and its metabolites, including catechol estrogens (CEs) and catechol estrogen quinones (CE-Qs) is closely related to breast cancer. Polymorphisms of the genes involved in the catechol estrogens metabolism pathway (CEMP) have been shown to affect the production of CEs and CE-Qs. In this study, we measured the induction of CYP1A1, CYP1B1, COMT, and GSTP1 by 17ß-estradiol (17ß-E2) in leukocytes with CYP1A1(∗)2C, CYP1B1(∗)3, COMT Val158Met and GSTP1 Ile105Val polymorphisms by semi quantitative RT-PCR and compared the values to those of leukocytes with wild type alleles; we also compared the differences in formation of 4- hydroxyestradiol (4-OHE2) and DNA-adducts. The data show that in the leukocytes with mutant alleles treatment with 17ß-E2 up-regulates CYP1A1 and CYP1B1 and down-regulates COMT mRNA levels, resulting in major increments in 4-OHE2 levels compared to leukocytes with wild-type alleles. Therefore, we propose induction levels of gene expression and intracellular 4-OHE2 concentrations associated with allelic variants in response to exposure of 17ß-E2 as a noninvasive biomarker that can help determine the risk of developing non-hereditary breast cancer in women.

Genetic susceptibility to lung cancer based on candidate genes in a sample from the Mexican Mestizo population: a case-control study.

BACKGROUND: Lung cancer (LC) is the leading cause of mortality caused by neoplasias worldwide. Although cigarette smoking is the primary cause, not all smokers develop LC. Polymorphic variations in genes associated with carcinogen metabolism, DNA repair, and cell-cycle dysregulation may alter an individual risk of developing LC. A polygenic cancer model was proposed, which considers genetic susceptibility to cancer is a global mechanism and suggests that it might be defined by the contributions of low-risk alleles in several candidate genes. This study focused on the analysis of 15 polymorphisms in 12 low-penetrance genes in a case-control study of a sample of Mexican Mestizo population. METHODS: A case-control study was performed with a total of 572 unrelated individuals, including 190 cases with a primary LC diagnosis and 382 healthy controls. The polymorphic status of the individuals was determined by TaqMan probe and RFLP techniques. The association between LC and genotype score (GS) was assessed by logistic regression. RESULTS: The results suggests a protective effect of the genotypes Arg/Lys of AhR rs2066853 (odds ratio [OR] 0.55, p = 0.03), Ile/Val of CYP1A1 rs1048943 (OR 0.49, p = 0.009), Tyr/His of EPHX1 rs1051740 (OR 0.53, p = 0.03), and A/A of CCND1 rs603965 (OR 0.44, p = 0.02). Analyses using the GS suggest that average cases have a larger number of risk alleles than controls (Student's t test -4.85, p = 0.001; OR 1.25, p < 0.001). CONCLUSIONS: Our results suggest significant differences between the GS for the cases and controls, which support the hypothesis underlying the additive and polygenic models for lung cancer risk depending on the polymorphisms in low-penetrance genes.

Polymorphisms of catechol estrogens metabolism pathway genes and breast cancer risk in Mexican women.

Breast cancer is associated to estrogen exposure. Allelic variants involved in estrogen metabolism might change the risk of developing this neoplasia. We examined the potential association of breast cancer risk in Mexican women with the polymorphisms CYP1A1 rs1048943, CYP1B1 rs1056836, COMT rs4680, GSTP1 rs1695, GSTT1 null and GSTM1 null which are involved in estrogen metabolism pathway. This study included 150 cases and 150 controls. A significant association was observed between, CYP1A1 rs1048943 (OR = 1.95, C.I. 1.13-3.36) and GSTP1 rs1695 (OR = 2.39, C.I. 1.24-4.24) polymorphisms with the risk of breast cancer. This risk was increased when the women were stratified according to their menopausal status. The results show that breast cancer risk significantly increases in women with 3-6 risk polymorphisms (OR = 3.75, C.I. 1.44-9.74).

Seasonal variations in the levels of PAH-DNA adducts in young adults living in Mexico City.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous components of polluted air. The Mexico City Metropolitan Area (MCMA), one of the most densely populated areas in the world, is 2240 m above sea level. At this altitude, less oxygen is available, making combustion less efficient and therefore producing more PAH pollutants. According to the Automatic Monitoring Network in Mexico City (RAMA, for its Spanish initials; http://www.sma.df.gob.mx/simat2/informaciontecnica/index.php?opcion=5&opciondifusion_bd=90), which performs environmental monitoring, the critical air pollutants in Mexico City are ozone and particulate matter (PM). PM emissions increase during the dry season (winter to spring) and decrease during the rainy season (summer to autumn). The bioactivation of some PAHs produces reactive metabolites that bind to DNA, and the presence of elevated levels of PAH-DNA adducts in tissues such as blood lymphocytes represents an elevated risk for the development of cancer. We have compared the levels of PAH-DNA adducts and the percentage of cells with chromosomal aberrations (CWAs) using a matched set of peripheral blood lymphocytes obtained on two separate occasions from young non-smoking inhabitants of the MCMA (n = 92) during the 2006 dry season and the following rainy season. PAH-DNA adducts were analysed using the r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)-DNA chemiluminescence immunoassay (CIA). The percentages of CWA were determined in cultured lymphocytes from the same individuals. Both DNA adduct levels and chromosomal aberrations were tested for correlation with lifestyle and the polymorphisms of cytochromes P450 CYP1A1 and CYP1B1 as well as glutathione-S-transferases GSTM1 and GSTT1. The levels of PAH-DNA adducts were significantly higher (P < 0.001) in the dry season (10.66 ± 3.05 per 10(9) nt, n = 92) than during the rainy season (9.50 ± 2.85 per 10(9) nt, n = 92) and correlated with the seasonal levels of particulate matter with a diameter of ≤ 10 µm (PM(10)). The percentage of CWA was not seasonally related; however, significant associations between the number of risk alleles and adduct levels in the dry (R = 0.298, P = 0.048) and in the wet seasons (R = 0.473, P = 0.001) were observed.

Polymorphism of CYP1A1*2C, GSTM1*0, and GSTT1*0 in a Mexican Mestizo population: a similitude analysis.

Cytochrome 1A1 (CYP1A1), glutathione transferase M1 (GSTM1), and glutathione transferase T1 (GSTT1) catalyze the bioactivation and detoxification of a wide variety of xenobiotic compounds that are mutagenic and/or carcinogenic (e.g., polycyclic aromatic hydrocarbons). Genetic polymorphisms of these metabolizing enzymes have been shown to affect individual susceptibility to environmental carcinogenic compounds. Although several studies have been published on the relationship between CYP1A1*2C, GSTM1*0, or GSTT1*0 polymorphism and cancer, not all findings can be extrapolated to other populations because of interethnic variability. Here, we investigate the frequency of CYP1A1*2C, GSTM1*0, or GSTT1*0 in a sample of Mexican Mestizos. We find that the frequency of GSTM1*0 is 0.335, that of GSTT1*0 is 0.121, and that of GSTM1*0 + GSTT1*0 is 0.023. The frequency of CYP1A1*2C is 0.54. Similitude analysis sets the Latin American populations in a common cluster near the Asian population, suggesting that the CYP1A1*2C polymorphism may have originated from this population and suffered a founder effect in the American population. Analysis of CYP1A1*2C, GSTM1*0, and GSTT1*0 haplotypes reveals that 35% of the population has some combination of risk genotypes. Taken together, these results point to a high susceptibility of the Mexican Mestizo population to the effects of environmental carcinogens.

Increased cytogenetic damage in outdoor painters.

Painters are exposed to an extensive variety of hazardous substances such as organic solvents, lead-containing pigments and residual plastic monomers. In this particular case, workers used commercially available exterior paints and occasionally gasoline or thinner as solvents. The application or removal of paints was performed without protection (masks or gloves). To determine occupational exposure risk, a monitoring study was designed. Group selection was made after a questionnaire administration, which included questions about lifestyle and medical history to exclude exposure to other potential sources of genotoxics. Smoking and drinking habits were also considered. Blood and buccal cell samples were obtained from 25 public building male painters and from a similar number of age- and gender-matched controls. Lead levels were measured in paint samples and in individuals' blood. Organic solvents and/or its metabolites were also determined in blood. Chromosomal aberrations (CA) and sister chromatid exchanges (SCE) were determined in peripheral blood lymphocyte cultures. Also, the frequency of micronuclei (MN) in buccal cells was investigated. Painters had higher lead levels in blood (p<0.05); CA and SCE in lymphocytes and MN in epithelial cells were also elevated (p<0.05). Cytogenetic damage was significantly associated with occupational exposure time but not with the levels of lead found in blood.

Changes in hemolymph glutathione status after variation in photoperiod and light-irradiance in crayfish Procambarus clarkii and Procambarus digueti.

This work studied the effect of light-stressors, irradiance and photoperiod length on the status of hemolymph glutathione in two species of crayfish, Procambarus clarkii and Procambarus digueti. Adult animals of each species were submitted to two experimental approaches: (1) two batches of each species were placed under low or high light irradiant conditions of light-dark (LD) 24 h cycles of two different photoperiod lengths, one normal LD 12: 12 and one extreme LD 20:4 low and high irradiance for 10 weeks. Time-dependent light changes on hemolymph glutathione concentration were determined throughout the entire experimental period; and (2) three batches of the two species were submitted to independent treatments consisting of the same LD 12:12 cycles of high and low irradiance and 20:4 high-irradiance LD cycles. Reduced and oxidized glutathione hemolymph concentrations were determined and total glutathione was calculated. In addition midgut glutathione reductase activity in both species was determined. The two species showed different hemolymph glutathione reactivity and glutathione status for the two light parameters. Dissimilar responses of both species, as well as the rate of mortality of P. digueti represent specific differences in the metabolic responses, as well as tolerance to photo-oxidative stress produced by light. The role of glutathione in the tolerance of crayfish to photo-oxidative stress is discussed.

Altered profile of urinary arsenic metabolites in adults with chronic arsenicism. A pilot study.

Relationships between alterations in the profile of urinary arsenic (As) species and the presence of cutaneous signs of arsenicism were studied in Region Lagunera, Mexico. The use of urinary concentrations of putative substrates and products of the As metabolism pathway, as indicators of metabolic efficiency is also discussed. Arsenic was determined by hydride generation atomic absorption spectrophotometry and separation of As species was performed by ion exchange chromatography. The exposed group had an average of 0.408 mg As/l of total As (TAs) in their drinking water, whereas "control' individuals had 0.031 mg/l. Urinary concentrations of arsenic species and TAs were 20 to 95 times higher in the exposed group. Significant increases in the relative proportions of inorganic arsenic (Asi) and monomethylarsonic acid (MMA), accompanied by decreases of dimethylarsinic acid (DMA) were also found in exposed individuals. Therefore, significant decreases in the value of the MMA/Asi, DMA/MMA and DMA/ Asi ratios were observed, suggesting a decreased As methylating ability. Exposed individuals bearing cutaneous signs had a significantly longer time of exposure, higher urinary concentrations and proportions of MMA and MMA/Asi values, and significantly lower DMA/ MMA than exposed individuals without cutaneous signs. Further research is needed to identify better parameters for assessing the efficiency of As metabolism in chronically exposed populations and to confirm the potential relationship between metabolic alterations and overt signs of As toxicity.

Genotoxic effects of metronidazole.

Metronidazole (MTZ) is an effective agent used in the treatment of parasitic infections. Its genotoxic effects have been shown in a variety of prokaryotic systems; however, negative results have been reported in human in vivo studies. Due to its wide spread use, a study was performed to evaluate the chromosomal aberration frequencies in peripheral blood lymphocyte cultures from 10 individuals, before and after metronidazole treatment. A significant increase in the percentage of cells with chromatid and isochromatid breaks was observed after metronidazole treatment (1500 mg per day for 10 days). The percentages of cells with aberrations did not correlate with the levels of MTZ found in plasma. Individual variability was observed with respect to both the induction of aberrations and the concentration of MTZ in plasma. They could represent differences at the metabolic level, since metronidazole is known to be biotransformed by a polymorphic P450 cytochrome, and its metabolites have shown mutagenic activity.

Environmental toxicants in developing countries.

Health effects from environmental toxicants may be a more serious problem in developing countries compared with developed countries because the problem is potentiated by other factors: a) the lack of or failure to enforce regulations, which allows human exposures to genotoxic agents; b) undernourishment of the lower economic and social classes that comprise the most exposed populations from industrial and agricultural activities; and c) parasitic infections that afflict a wide range of populations in both urban and rural areas. Data on the genotoxic effects of different types of exposures, including environmental exposes (natural and industrial), occupational exposures, and infections and medical treatments, are presented and discussed with the point of view that all these factors must be taken into account with respect to regulation and the protection of human health. Occupational exposures in developing countries are higher than in developed countries due to lack of stringent regulations, lack of knowledge of the risks involved, and the negligence of workers. General pollution is another important issue since developed countries have established strict regulations and risky industrial processes are being exported to developing countries, along with banned substances and dangerous industrial wastes. It should be emphasized that stringent regulations in developed countries will not prevent exposures in the long term because toxic substances that are released into the environment will ultimately reach all our future generations.

Sodium 2,3-dimercaptopropane-1-sulfonate challenge test for mercury in humans. III. Urinary mercury after exposure to mercurous chloride.

The sodium salt of 2,3-dimercaptopropane-1-sulfonic acid (Dimaval; DMPS) challenge test has been given previously to humans exposed to elemental mercury (vapor) or mercuric salts, but not mercurous salts. The test (300 mg p.o., after an 11-hr fast) was given to 11 factory workers who make a skin lotion that contains mercurous chloride, eight users of the skin lotion and nine controls. Urines were analyzed for total mercury by using cold vapor atomic absorption spectrophotometry. The mercury excreted for 6 hr before and 6 hr after DMPS treatment was 113 micrograms +/- 26 and 5037 micrograms +/- 682 S.E.M. for the skin lotion makers; 16.2 micrograms +/- 3.4 and 1410 micrograms +/- 346 S.E.M. for the skin lotions users; and 0.49 micrograms +/- 0.11 and 18.4 micrograms +/- 7.1 S.E.M. for the controls, respectively. The increases in urinary mercury resulting from the DMPS challenge test were 45-, 87- and 38-fold, respectively. The results demonstrate that, in humans exposed to mercurous chloride, DMPS increases the urinary excretion of mercury and that the DMPS/mercury challenge test is of value for a more realistic estimation of mobilizable mercury. An attempt to associate genotoxicity, as indicated by micronuclei content in buccal cells, with mercury exposure was inconclusive, perhaps because of the small number of subjects.

Aneugenic effect of sodium arsenite on human lymphocytes in vitro: an individual susceptibility effect detected.

Arsenic is a well known carcinogenic environmental pollutant although its mechanism of action remains unknown. Since alterations in chromosome segregation have been observed in individuals exposed to high concentrations of arsenic in the drinking water, the aneuploidogenic potential of arsenic was evaluated in vitro. Whole blood cultures were incubated for 72 h and treated with various concentrations of sodium arsenite for the last 24 h. Cells were harvested and samples were processed specially for aneuploidy evaluation. The number of chromosomes in 200 metaphases of first and second division cells was scored. A dose-related effect was observed: the highest concentration (10(-2) microM) induces 28.33% and 22.4% hyperploid cells in first and second division respectively and 29% tetraploid cells. The colchicine-like effect of arsenic was also evaluated. Mitotic arrest was evaluated in cultures treated for the last 2 h. Sodium arsenite can produce 40.24% and 12.93% of the colcemid effect (mitotic arrestant effect at 10(-2) microM and 10(-10) microM respectively). A different individual susceptibility effect was observed in both parameters and confirmed with the chromosome aberrations levels induced by arsenic in the same donors. Data indicate that sodium arsenite has an aneuploidogenic and a mitotic arrestant effect.

Genotoxic monitoring of workers at a hazardous waste disposal site in Mexico.

Chromosomal aberration and sister chromatid exchange (SCE) frequencies were determined in lymphocytes cultured from 12 high-risk individuals working at a landfill for hazardous waste disposal. Cell proliferation kinetics (CPK) was also determined. Compared with 7 control individuals, no effects were observed with respect to SCE nor on CPK. However, the workers exhibited significantly higher frequencies of chromatid and chromosomal deletions, the magnitude of which was related with exposure time. This study suggests that when high-risk exposure is suspected, determining biomarkers of genotoxic damage (e.g., chromosomal aberrations), is useful for risk assessments.

Altered urinary porphyrin excretion in a human population chronically exposed to arsenic in Mexico.

1. A detailed study of the urinary excretion pattern of porphyrins in humans chronically exposed to As via drinking water was performed using high performance liquid chromatography (HPLC) 2. Thirty-six individuals (15 men and 21 women) were selected from a town which had 0.400 mg L-1 of As in drinking water. The control group consisted of thirty-one individuals (13 men and 18 women) whose As concentration in drinking water was 0.020 mg L-1. 3. The major abnormalities in the urinary porphyrin excretion pattern observed in arsenic-exposed individuals were: (a) significant reductions in coproporphyrin III excretion resulting in decreases in the COPRO III/COPRO I ratio, and (b) significant increases in uroporphyrin excretion. Both alterations were responsible for the decrease in the COPRO/URO ratio. 4. No porphyrinogenic response was found in individuals with urinary As concentrations below 1,000 micrograms of As g-1 of creatinine. However, as arsenic concentrations exceeded this value, the excretion of porphyrins (except coproporphyrin III) increased proportionally. 5. The prevalence of clinical signs of arsenicism showed a direct relationship to both As concentration in urine and time-weighted exposure to As. A direct relationship between time-weighted exposure and alterations in urinary porphyrin excretion ratios was also observed. 6. The alterations found are compatible with a lower uroporphyrinogen decarboxylase activity in arsenic-exposed individuals. However, the similarities in the urinary porphyrin excretion pattern between As-exposed individuals and Dubin-Johnson syndrome patients suggest that impairments in the excretion of coproporphyrin isomers may also contribute to the pattern observed.

Mean dose to lymphocytes during radiotherapy treatments.

Using a probabilistic model with parameters from four radiotherapy protocols used in Mexican hospitals for the treatment of cervical cancer, we have calculated the distribution of dose to cells in peripheral blood of patients. Values of the mean dose to the lymphocytes during and after a 60Co treatment are compared to estimates from an in vivo chromosome aberration study performed on five patients. Calculations indicate that the mean dose to the circulating blood is about 2% of the tumor dose, while the mean dose to recirculating lymphocytes may reach up to 7% of the tumor dose. Differences up to a factor of two in the dose to the blood are predicted for different protocols delivering equal tumor doses. The data suggest mean doses higher than the predictions of the model.

Lymphocyte replicating ability in individuals exposed to arsenic via drinking water.

A human monitoring study was carried out to explore the effect on lymphocyte proliferation of chronic exposure to arsenic (As) via drinking water. Blood and urine samples were taken from volunteers from a town where levels of As in the drinking water averaged 412 micrograms/l, and from a matched group of individuals, with similar socioeconomic status, that drank water with As average levels of 37.2 micrograms/l. Exposure was assessed by questionnaires and by determining the levels of As in urine and water samples. The evaluation of the peripheral blood lymphocyte proliferation was done at different culture times using labelling (LI), mitotic (MI) and replication indexes (RI) as endpoints. No significant differences were seen for either LI or MI, except for MI in 72 h cultures and in LI in males and females with skin lesions vs. those without lesions. Significant differences in RI were seen for exposed females but not for males. Correlations between LI and MI showed that progression from the initial S-to M-phase is altered in exposed individuals. Arsenic exposure as well as lead and mercury affect cellular immune response, making the endpoints of cell proliferation variables of interest in population monitoring study design, since they might provide information in health impairment due to exposure, which is important in risk assessment.

Evaluation of the carcinogenic and genotoxic potential of praziquantel in the Syrian hamster embryo cell transformation assay.

Praziquantel, a drug used for the treatment of neurocysticercosis, was tested for its ability to induce morphological transformation of Syrian hamster embryo fibroblasts. Results indicate that praziquantel transforms these cells without affecting their viability. Further experiments were carried out to investigate its possible mechanism of action in the same cell system. Micronucleus formation was observed in cultures treated with concentrations which induced morphological transformation, about 40% of these micronuclei were positive to a kinetochore antibody. No induction of DNA repair synthesis was observed even at cytotoxic concentrations. These results suggest that praziquantel has an aneugenic effect which could be responsible for its ability to transform morphologically these cells. Risk-benefit analysis should be carried out whenever this drug is utilized.

Mitotic index and cell proliferation kinetics for identification of antineoplastic activity.

The mitotic index (MI) and cell proliferation kinetics (CPK) of human blood lymphocyte cultures were determined to evaluate the effects of six antineoplastic drugs with well known cytostatic activity: cisplatin, melphalan, bleomycin, methotrexate, 5-fluorouracil and 6-mercaptopurine. All six drugs showed a clear effect on the inhibition of MI. The first three drugs interact directly with DNA showing a dose-related retardation of CPK. Methotrexate, 5-fluorouracil and 6-mercaptopurine, which act on ribonucleotide biosynthesis, showed no significant effects on CPK. The results suggest that CPK and MI measurements are useful for the prescreening of drugs with potential cytostatic activity.