María E. Gonsebatt

The role of antioxidants and antioxidant-related enzymes in protective responses to environmentally induced oxidative stress

In aerobic organisms, oxygen is essential for efficient energy production but paradoxically, produces chronic toxic stress in cells. Diverse protective systems must exist to enable adaptation to oxidative environments. Oxidative stress (OS) results when production of reactive oxidative species (ROS) exceeds the capacity of cellular antioxidant defenses to remove these toxic species. Epidemiological and clinical studies have linked environmental factors such as diet and lifestyle to cancer, diabetes, atherosclerosis, and neurodegenerative disorders. All of these conditions, as well as the aging process, are associated with OS due to elevation of ROS or insufficient ROS detoxification. Many environmental pollutants engage signaling pathways that are activated in response to OS. The same sequences of events are also associated with the etiology and early pathology of many chronic diseases. Investigations of oxidative responses in different in vivo models suggest that, in complex organisms such as mammals, organs and tissues contain distinct antioxidant systems, and this may form the basis for differential susceptibility to environmental toxic agents Thus, understanding the pathways leading to the induction of antioxidant responses will enable development of strategies to protect against oxidative damage. We shall review evidence of organ-specific antioxidant responses elicited by environmental pollutants in humans and animal models.

Cytogenetic effects in human exposure to arsenic

The cytogenetic effects of arsenic exposure were studied among rural populations that live in the same geographical area and have similar socioeconomic status, but different degree of exposure to inorganic arsenic (As) via drinking water. A group of inhabitants of Santa Ana (408.17 micrograms/l of As in drinking water) were considered the exposed individuals and a group of inhabitants of Nazareno (29.88 micrograms/l) were considered as controls. Blood and urine samples were obtained from volunteers. Past and current exposure, health, and nutritional status as well as the presence of arsenic skin lesions were ascertained in study participants through questionnaires and physical examination. The frequencies and types of chromosomal aberrations in first-division metaphases were studied in whole blood lymphocyte cultures while the presence of micronuclei (MN) was studied in exfoliated epithelial cells obtained from the oral mucosa and from urine samples. Total arsenic (TAs) content, and the relative proportions of inorganic arsenic (IAs), and the metabolites monomethylarsonic (MMA) and dimethylarsinic (DMA) acid were determined in urine samples. Exposed individuals showed a significant increase in the frequency of chromatid and isochromatid deletions in lymphocytes and of MN in oral and urinary epithelial cells. Males were more affected than females, and a higher number of micronucleated oral cells were found among those individuals with skin lesions. The type of cytogenetic damage observed gives evidence of arsenic as a clastogenic/aneugenic carcinogen.

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.

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.

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.

Altered Urinary Porphyrin Excretion in a Human Po p ulation 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 μg 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.

Inorganic arsenic effects on human lymphocyte stimulation and proliferation

Lymphocyte cultures from individuals exposed to high levels of hydroarsenicism showed a slower cell cycle kinetics than cultures from low-exposed individuals. Since this difference in proliferation could be due to chronic arsenic exposure, the in vitro effects of inorganic arsenic in human whole blood lymphocyte cultures were investigated. When lymphocytes were exposed to concentrations of arsenite and arsenate similar to those found in the blood of exposed subjects (10(-7), 10(-8) and 10(-9) M) during the last 24 h before harvesting, a dose-related inhibition of proliferation was observed. Cultures were also treated with 10(-9) M of arsenite and arsenate for 2, 6 and 24 h at the beginning of the cultures in the presence or absence of phytohemagglutinin (PHA). Inhibition of stimulation and proliferation was directly related to the length of treatment. The results show that, at the concentrations tested, arsenite and arsenate impair lymphocyte stimulation and proliferation and confirm the fact that chronic arsenic exposure can affect the proliferation of whole blood lymphocytes.

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.

Arsenic species, AS3MT amount, and AS3MT gen expression in different brain regions of mouse exposed to arsenite

Human exposure to inorganic arsenic (iAs) has been associated with cancer and serious injury to various internal organs, as well as peripheral neuropathy, endocrine disruption and diverse effects in the central nervous system (CNS). Using rodent models, it is possible to demonstrate As accumulation in the brain that leads to defects in operant learning, behavioral changes, and affect pituitary gonadotrophins. iAs biomethylation in the CNS is a significant process, yielding products that are more reactive and toxic than the parent compound. Mice received 2.5, 5, and 10 mg/kg/day sodium arsenite orally for 9 days. We investigated the distribution of iAs and its metabolites as well as the mRNA and protein expression of arsenic (III) methyltransferase (AS3MT), which encodes the key enzyme in iAs metabolism, in the cerebral cortex, hippocampus, striatum, mesencephalon, thalamus, cerebellum, hypothalamus, pons, medulla oblongata, and pituitary of mouse brain. Our findings show that methylated As metabolites are present in all brain regions studied suggesting that AS3MT is ubiquitously expressed in the brain and it is not inducible by dose of arsenite. There is also a dose-related accumulation of As species in all brain regions, with the highest accumulation observed in the pituitary. The higher distribution of arsenicals in pituitary can help to explain the neuroendocrine effects associated with iAs exposure.

Are mitotic index and lymphocyte proliferation kinetics reproducible endpoints in genetic toxicology testing

Lymphocyte proliferation kinetics is an endpoint used in genetic toxicology which has recently been proposed as an alternative for the screening of new cytostatic drugs. Although great variability for this parameter has been reported, there are few reports about the intra- and inter-individual variation of the effects of chemicals on this endpoint. For this reason, experiments were conducted to evaluate the reproducibility of the effects of a well-known cytostatic, mitomycin C (MMC), on the proliferation of PHA-stimulated human lymphocytes, both over time and among samples from several donors. Although inter-individual variability was shown in both parameters in untreated and treated cultures, this variation was not significant. Intra-individual variation was significantly detected only in cultures treated with 0.1 microM MMC.