Impact of Cadmium Mediated by Tobacco Use in Musculoskeletal Diseases.

Tobacco use has a negative impact on health due to its relationship with the development of high-mortality diseases, such as pulmonary cancer. However, the effect of cadmium (Cd), present in tobacco smoke, on the development of joint diseases has been scarcely studied. The objective of this review is to discuss the evidence regarding the mechanisms by which Cd exposure, through tobacco smoke, may lead to the development of osteoarthritis (OA), osteoporosis (OP), and rheumatoid arthritis (RA). There's evidence suggesting a string association between moderate to severe OA development and tobacco use, and that a higher blood concentration of Cd can trigger oxidative stress (OS) and inflammation, favoring cartilage loss. At the bone level, the Cd that is inhaled through tobacco smoke affects bone mineral density, resulting in OP mediated by a decrease in the antioxidant enzymes, which favors the bone resorption process. In RA, tobacco use promotes the citrullination process through Cd exposure and increases OS and inflammation. Understanding how tobacco use can increase the damage at the articular level mediated by a toxic metal, i.e., Cd, is important. Finally, we propose prevention, control, and treatment strategies for frequently disabling diseases, such as OA, OP, and RA to reduce its prevalence in the population.

Effect of cadmium on the concentration of essential metals in a human chondrocyte micromass culture.

BACKGROUND: An essential element imbalance in the joint might favor gradual degeneration of the articular cartilage. It has been reported that cadmium (Cd) plays an antagonistic role with regards to the presence of essential elements, such as zinc (Zn), iron (Fe), and manganese (Mn), which may favor the development of disabling diseases, like osteoarthritis (OA) and osteoporosis. METHODS: 3D cultures of human chondrocytes were phenotyped with the Western blot technique and structurally evaluated with histological staining. The samples were exposed to 1, 5, and 10 µM of CdCl2 for 12 h, with a non-exposed culture as control. The concentration of Cd, Fe, Mn, Zn, chromium (Cr), and nickel (Ni) was quantified through plasma mass spectrometry (ICP-MS). The data were analyzed with a Kruskal Wallis test, a Kendall's Tau test and Spearman's correlation coefficient with the Stata program, version 14. RESULTS: Our results suggest that Cd exposure affects the structure of micromass cultures and plays an antagonistic role on the concentration of essential metals, such as Zn, Ni, Fe, Mn, and Cr. CONCLUSION: Cd exposure may be a risk factor for developing joint diseases like OA, as it can interfere with cartilage absorption of other essential elements that maintain cartilage homeostasis.

Prenatal exposure to metals modified DNA methylation and the expression of antioxidant- and DNA defense-related genes in newborns in an urban area.

The developmental period in utero is a critical window for environmental exposure. Epigenetic fetal programming via DNA methylation is a pathway through which metal exposure influences the risk of developing diseases later in life. Genetic damage repair can be modified by alterations in DNA methylation, which, in turn, may modulate gene expression due to metal exposure. We investigated the impact of prenatal metal exposure on global and gene-specific DNA methylation and mRNA expression in 181 umbilical cord blood samples from newborns in Mexico City. Global (LINE1) and promoter methylation of DNA-repair (OGG1 and PARP1) and antioxidant (Nrf2) genes was evaluated by pyrosequencing. Prenatal metal exposure (As, Cu, Hg, Mn, Mo, Pb, Se, and Zn) was determined by ICP-MS analysis of maternal urine samples. Multiple regression analyses revealed that DNA methylation of LINE1, Nrf2, OGG1, and PARP1 was associated with potentially toxic (As, Hg, Mn, Mo, and Pb) and essential (Cu, Se, and Zn) elements, and with their interactions. We also evaluated the association between gene expression (mRNA levels quantified by p-PCR) and DNA methylation. An increase in OGG1 methylation at all sites and at CpG2, CpG3, and CpG4 sites was associated with reduced mRNA levels; likewise, methylation at the CpG5, CpG8, and CpG11 sites of PARP1 was associated with reduced mRNA expression. In contrast, methylation at the PARP1 CpG7 site was positively associated with its mRNA levels. No associations between Nrf2 expression and CpG site methylation were observed. Our data suggest that DNA methylation can be influenced by prenatal metal exposure, which may contribute to alterations in the expression of repair genes, and therefore, result in a lower capacity for DNA damage repair in newborns.

Effects of inorganic arsenic exposure on glucose transporters and insulin receptor in the hippocampus of C57BL/6 male mice.

Children and adolescent populations chronically exposed to high doses of inorganic arsenic (iAs) in drinking water in some regions around the world have shown behavioral and memory deficits. Recent studies have also associated iAs exposure with dysregulation of glucose metabolism. The hippocampus is a cerebral region well known for its role in learning and memory. Studies in vitro and in vivo have shown that the hippocampus is vulnerable to iAs exposure, and to changes in glucose metabolism. The glucose transporters (GLUTs) and insulin receptor (IR) regulate glucose metabolism in brain; they are expressed by hippocampal cells, and alterations in these proteins have been associated with memory deficits. The aims of this study were to evaluate the effects of iAs exposure via drinking water (DW) on GLUT1, GLUT3 and insulin receptor (INSR) mRNA expression in the hippocampus, on performance in a spatial memory task, and on peripheral glucose regulation. C57Bl/6 male mice were exposed to 50 mg iAs/L via DW for one, two, or three months. The qRT-PCR analyses indicated that, compared to a control group, GLUT1 and GLUT3 mRNA levels were decreased, while INSR mRNA levels were increased in the hippocampus of iAs exposed animals. The levels of iAs and its methylated species in the hippocampus of the iAs-exposed group were significantly higher than in controls. Mice exposed to iAs learned the spatial task but showed increased latency to find the submerged platform 48 h after the last training session; these animals also showed dysregulation of peripheral glucose. These results suggest that the effects of iAs exposure on a spatial memory task performance could be mediated by disruptions of glucose regulation in the CNS.

Nuclear factor erythroid 2-related factor gene variants and susceptibility of arsenic-related skin lesions.

Inorganic arsenic (iAs) is an important pollutant associated with various chronic-degenerative diseases. The cytoprotective protein nuclear factor erythroid 2-related factor (NRF2) has been proposed as an important responsive mechanism against iAs exposure. The aim of this study was to determine whether the risk of skin lesions in people exposed to iAs-contaminated water could be modified by the presence of single nucleotide polymorphisms in the NRF2 coding gene. We studied 117 individuals with long-term iAs exposure and 120 nonexposed individuals. Total As was determined in water, meanwhile iAs and its metabolites were measured in urine. The iAs-induced skin lesion status was evaluated by expert dermatologists. We sequenced the promoter region of NRF2 in a sample of 120 healthy donors. We found four polymorphisms previously reported and one novel polymorphism in the 5' regulatory region of the NRF2. In this study, we did not find allelic and genotype association of NRF2 polymorphisms with iAs-related skin lesion. However, the analysis of haplotypes composed by -653GA, and -617CA NRF2 single nucleotide polymorphisms showed a significant association with protection against skin lesions in the low-As exposure group. This is the first report studying the association between NRF2 polymorphisms and susceptibility of As-related skin lesions. Increasing the sample size will allow us to confirm this data.

Association of glutathione S-transferase Ω 1-1 polymorphisms (A140D and E208K) with the expression of interleukin-8 (IL-8), transforming growth factor beta (TGF-ß), and apoptotic protease-activating factor 1 (Apaf-1) in humans chronically exposed to arsenic in drinking water.

Human exposure to arsenicals is associated with inflammatory-related diseases including different kinds of cancer as well as non-cancerous diseases like neuro-degenerative diseases, atherosclerosis, hypertension, and diabetes. Interindividual susceptibility has been mainly addressed by evaluating the role of genetic polymorphism in metabolic enzymes in inorganic arsenic (iAs) metabolism. Glutathione S-transferase omega 1-1 (GSTO1-1), which had been associated with iAs metabolism, is also known to participate in inflammatory and apoptotic cellular responses. The polymorphism A140D of GSTO1-1 has been not only associated with distinct urinary profile of arsenic metabolites in populations chronically exposed to iAs in drinking water, but also with higher risk of childhood leukemia and lung disease in non-exposed populations, suggesting that GSTO1-1 involvement in other physiologic processes different from toxics metabolism could be more relevant than is thought. We evaluated the association of the presence of A140D and E208K polymorphisms of GSTO1-1 gene with the expression of genes codifying for proteins involved in the inflammatory and apoptotic response in a human population chronically exposed to iAs through drinking water. A140D polymorphism was associated with higher expression of genes codifying for IL-8 and Apaf-1 mainly in heterozygous individuals, while E208K was associated with higher expression of IL-8 and TGF- gene, in both cases, the association was independently of iAs exposure level; however, the exposure to iAs increased slightly but significantly the influence of A140D and E208K polymorphisms on such genes expression. These results suggest an important role of GSTO1-1 in the inflammatory response and the apoptotic process and indicate that A140D and E208K polymorphisms could increase the risk of developing inflammatory and apoptosis-related diseases in As-exposed populations.

The effects of fluoride on cell migration, cell proliferation, and cell metabolism in GH4C1 pituitary tumour cells.

The consumption of drinking water rich in fluoride has toxic effects on the central nervous system. In cell biology research, fluoride is currently used as a phosphatase inhibitor. The aim of the present study was to evaluate the effect of fluoride on different physiological processes in GH4C1 pituitary tumour cells. We used a range of different fluoride concentrations, from levels below normal human serum concentrations (0.23 and 1.2 micromol/L) to those observed in chronically exposed persons (10.7 micromol/L) and above (107 and 1072 micromol/L). Treatment of 10.7 micromol/L fluoride resulted in a discrete induction of DNA synthesis, without a change in cell number. Cell migration, a behaviour stimulated by growth factors, was increased in cells treated with 2.4 micromol/L. At this fluoride concentration, changes in phosphorylation status of both cytoskeletal and cytosolic protein fractions, as well as in actin cytoskeletal arrangements were observed. The GH4C1 fluoride treated cells had significantly less cellular protein than control cells, suggesting an effect of fluoride on hormone secretion and protein synthesis in this endocrine cell. The bioreduction of MTT was significantly increased with a wide range of fluoride concentrations. With the highest fluoride concentration, 1072 micromol/L, all of the analysed parameters were significantly reduced, suggesting that this dose is highly toxic in GH4C1 cells. Our results show that biologically relevant concentrations of fluoride are capable of increasing cell migration in tumour cells, suggesting that exposure to fluoride could stimulate tumour invasion.

Effect of dietary selenium deficiency on the in vitro fertilizing ability of mice spermatozoa.

Selenium is an essential micronutrient for mammals, being integral part of antioxidant system. The aim of the study was to evaluate the effect of selenium deficiency on in vitro fertilization (IVF) capacity of spermatozoa and on oxidative stress in these cells. Male C57BL/6N mice were maintained on selenium-deficient or selenium-sufficient diets (0.02 or 0.2 ppm of selenium as selenomethionine, respectively) for 4 months. Liver glutathione peroxidase activity measurements were used to confirm selenium deficiency. Sperm quality and IVF capability among both groups were evaluated. To assess oxidative damage, lipid peroxidation as malondialdehyde production was determined in spermatozoa as well as the testes. Ultrastructural analyses of spermatozoa nuclei using transmission electron microscopy were also performed. The percentage of eggs fertilized with sperm from selenium-deficient mice was significantly decreased by approximately 67%. This reduced fertilization capacity was accompanied by increased levels of lipid peroxidation in both the testes and sperm, indicating that selenium deficiency induced oxidative stress. Consistent with this finding, spermatozoa from selenium-deficient animals exhibited altered chromatin condensation. Deficiency in dietary selenium decreases the reproductive potential of male mice and is associated with oxidative damage in spermatozoa.

Arsenite induced oxidative damage in mouse liver is associated with increased cytokeratin 18 expression.

Cytokeratins (CK) constitute a family of cytoskeletal intermediate filament proteins that are typically expressed in epithelial cells. An abnormal structure and function are effects that are clearly related to liver diseases as non-alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. We have previously observed that sodium arsenite (SA) induced the synthesis of CK18 protein and promotes a dose-related disruption of cytoplasmic CK18 filaments in a human hepatic cell line. Both abnormal gene expression and disturbance of structural organization are toxic effects that are likely to cause liver disease by interfering with normal hepatocyte function. To investigate if a disruption in the CK18 expression pattern is associated with arsenite liver damage, we investigated CK18 mRNA and protein levels in liver slices treated with low levels of SA. Organotypic cultures were incubated with 0.01, 1 and 10 microM of SA in the absence and presence of N-acetyl cysteine (NAC). Cell viability and inorganic arsenic metabolism were determined. Increased expression of CK18 was observed after exposure to SA. The addition of NAC impeded the oxidative effects of SA exposure, decreasing the production of thiobarbituric acid-reactive substances and significantly diminishing the up regulation of CK18 mRNA and protein. Liver arsenic levels correlated with increased levels of mRNA. Mice treated with intragastric single doses of 2.5 and 5 mg/kg of SA showed an increased expression of CK18. Results suggest that CK18 expression may be a sensible early biomarker of oxidative stress and damage induced by arsenite in vitro and in vivo. Then, during SA exposure, altered CK expression may compromise liver function.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in mice following acute oral administration of arsenate.

The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or by parent chemical and metabolite acting together. This has emerged as an issue for inorganic arsenic (iAs), because both its trivalent and pentavalent methylated metabolites have unique toxicities; the methylated trivalent metabolites also exhibit greater potency than trivalent inorganic arsenic (arsenite, As(III)) for some endpoints. In this study, the time-course tissue distributions for iAs and its methylated metabolites were determined in blood, liver, lung, and kidney of female B6C3F1 mice given a single oral dose of 0, 10, or 100 micromol As/kg (sodium arsenate, As(V)). Compared to other organs, blood concentrations of iAs, mono- (MMA), and dimethylated arsenic (DMA) were uniformly lower across both dose levels and time points. Liver and kidney concentrations of iAs were similar at both dose levels and peaked at 1 h post dosing. Inorganic As was the predominant arsenical in liver and kidney up to 1 and 2 h post dosing, with 10 and 100 micromol As/kg, respectively. At later times, DMA was the predominant metabolite in liver and kidney. By 1 h post dosing, concentrations of MMA in kidney were 3- to 4-fold higher compared to other tissues. Peak concentrations of DMA in kidney were achieved at 2 h post dosing for both dose levels. Notably, DMA was the predominant metabolite in lung at all time points following dosing with 10 micromol As/kg. DMA concentration in lung equaled or exceeded that of other tissues from 4 h post dosing onward for both dose levels. These data demonstrate distinct organ-specific differences in the distribution and methylation of iAs and its methylated metabolites after exposure to As(V) that should be considered when investigating mechanisms of arsenic-induced toxicity and carcinogenicity.

Effects of arsenite on cell cycle progression in a human bladder cancer cell line.

Bladder cancer is one of the most important diseases associated with arsenic (As) exposure in view of its high prevalence and mortality rate. Experimental studies have shown that As exposure induces cell proliferation in the bladder of sodium arsenite (iAsIII) subchronically treated mice. However, there is little available information on its effects on the cell cycle of bladder cells. Thus, our purpose was to evaluate the effects of iAsIII on cell cycle progression and the response of p53 and p21 on the human-derived epithelial bladder cell line HT1197. iAsIII treatment (1-10 microM) for 24 h induced a dose-dependent increase in the proportion of cells in S-phase, which reached 65% at the highest dose. A progressive reduction in cell proliferation was also observed. BrdU was incorporated to cellular DNA in an interrupted form, suggesting an incomplete DNA synthesis. The time-course of iAsIII effects (10 microM) showed an increase in p53 protein content and a transient increase in p21 protein levels accompanying the changes in S-phase. These effects were correlated with iAs concentrations inside the cells, which were not able to metabolize inorganic arsenic. Our findings suggest that p21 was not able to block CDK2-cyclin E complex activity and was therefore unable to arrest cells in G1 allowing their progression into the S-phase. Further studies are needed to ascertain the mechanisms underlying the effects of iAsIII on the G1 to S phase transition in bladder cells.

Glutathione reductase inhibition and methylated arsenic distribution in Cd1 mice brain and liver.

Inorganic arsenic exposure via drinking water has been associated with cancer and serious injury in various internal organs, as well as with peripheral neuropathy and diverse effects in the nervous system. Alterations in memory and attention processes have been reported in exposed children, whereas adults acutely exposed to high amounts of inorganic arsenic showed impairments in learning, memory, and concentration. Glutathione (GSH) is extensively involved in the metabolism of inorganic arsenic, and both arsenite and its methylated metabolites have been shown to be potent inhibitors of glutathione reductase (GR) in vitro. Brain would be more susceptible to GR inhibition because of the decreased activities of superoxide dismutase (SOD) and catalase reported in this tissue. To investigate whether GR inhibition could be documented in vivo, we determined the activity and levels of GR in brain as well as in liver, the main organ of arsenic metabolism in mice exposed to 2.5, 5, or 10 mg/kg/day of sodium arsenite over a period of 9 days. In contrast to what has been observed in vitro, significant inhibition of the expression and activity of GR was observed only at the highest concentration used (10 mg/kg/day) in both organs. Although the disposition of arsenicals was higher in liver, significant amounts of inorganic and methylated arsenic forms were determined in the brain of exposed animals. The formation of monomethylarsenic (MMA) and dimethylarsenic (DMA) metabolites in the brain was confirmed by incubating brain slices for 24, 48, and 72 h with sodium arsenite.

Arsenic levels in cooked food and assessment of adult dietary intake of arsenic in the Region Lagunera, Mexico.

The aim of this paper is to estimate the levels of arsenic (As) ingestion through cooked foods consumed in an arsenic endemic area and the assessment of their dietary intake of As. The study was conducted in two villages: a population chronically exposed to a high concentration of As via drinking water (410+/-35 microg/l) and to a low-exposure group (12+/-4 microg/l). A 24-h dietary recall questionnaire was applied to about 25 adult participants in each community. Samples of cooked food, ready for intake, were collected separately from each family's participants. To obtain the As estimate for each food item consumed, the mean quantity of food ingested in grams (wet weight) was calculated and the concentrations of total arsenic (TAs) in each cooked food were determined. The estimations of TAs intake were based on the sum over mean of As ingested from each food item consumed during the 24-h period for each participant. For the estimation of total daily As intake, we summed the mean obtained from food, plain water and hot beverage intakes. The TAs average intakes calculated for low-As-exposure group were 0.94 and 0.76 microg/kg body weight/day, for both summer and winter exposure scenarios, respectively. These values are 44.7 and 36% of the provisional tolerable daily intake (PTDI) for inorganic arsenic (2.14 microg/kg body weight/day), established by the World Health Organization (WHO) in 1989. The WHO reference value was obtained on a weekly basis intake estimation assuming an average body weight of 68 kg in adults. In contrast, for the high-exposure group the TAs average intakes were 16.6 and 12.3 microg/kg body weight/day for summer and winter, respectively. Ingestion via cooked food represented 32.5 and 43.9% of the total daily As intake in the high-exposure group; for summer and winter, respectively. None the less, the bioavailability of As through food can be different than via drinking water.

Stress proteins induced by arsenic.

The elevated expression of stress proteins is considered to be a universal response to adverse conditions, representing a potential mechanism of cellular defense against disease and a potential target for novel therapeutics. Exposure to arsenicals either in vitro or in vivo in a variety of model systems has been shown to cause the induction of a number of the major stress protein families such as heat shock proteins (Hsp). Among them are members with low molecular weight, such as metallotionein and ubiquitin, as well as ones with masses of 27, 32, 60, 70, 90, and 110 kDa. In most of the cases, the induction of stress proteins depends on the capacity of the arsenical to reach the target, its valence, and the type of exposure, arsenite being the biggest inducer of most Hsp in several organs and systems. Hsp induction is a rapid dose-dependent response (1-8 h) to the acute exposure to arsenite. Thus, the stress response appears to be useful to monitor the sublethal toxicity resulting from a single exposure to arsenite. The present paper offers a critical review of the capacity of arsenicals to modulate the expression and/or accumulation of stress proteins. The physiological consequences of the arsenic-induced stress and its usefulness in monitoring effects resulting from arsenic exposure in humans and other organisms are discussed.

Alloxan decreases intracellular potassium content of the isolated frog skin epithelium.

Alloxan has been widely used to provoke diabetes mellitus. This compound induces necrosis of the beta-pancreatic cells and the renal tubules. However, the mechanism of this action has not been fully established. There is some evidence that this drug may act by an alteration of several ionic transport mechanisms. Nevertheless, there is scant information on the effect of alloxan on these ionic transport mechanisms of the membrane in epithelial cells. We reported that this drug induces a decrease in sodium transport in the frog skin. In order to obtain information about the mechanism involved in the sodium transport diminution provoked by alloxan, in this study the function of Na+-K+ ATPase enzyme on transepithelial sodium transport altered by alloxan is explored. We measured changes in the short circuit current and in the intracellular content of sodium and potassium under conditions of maximally stimulated enzyme activity. Short circuit current was not modified by the treatment with alloxan during the period of highest activity of the enzyme, suggesting a site of action independent of this ATPase. Cell potassium was reduced in alloxan-treated epithelia, without significant changes in Na+ content. This finding points out the existence of an alteration induced by alloxan of some modulator mechanisms of the intracellular K+ concentration. The treatment of the frog skin with cesium chloride, a K+ channel blocker, prevented the decrease of Na+ transport produced by alloxan. This result suggests an action of this diabetogenic drug on the K+ channels of the frog skin epithelium.

Determination of trivalent methylated arsenicals in biological matrices.

The enzymatically catalyzed oxidative methylation of As yields methylated arsenicals that contain pentavalent As (As(V)). Because trivalent As (As(III)) is the favored substrate for this methyltransferase, methylated arsenicals containing As(V) are reduced to trivalency in cells. Methylated arsenicals that contain As(III) are extremely potent inhibitors of NADPH-dependent flavoprotein oxidoreductases and potent cytotoxins in many cell types. Therefore, the formation of methylated arsenicals that contain As(III) may be properly regarded as an activation step, rather than a means of detoxification. Recognition of the role of methylated arsenicals that contain As(III) in the toxicity and metabolism of As emphasizes the need for analytical methods to detect and quantify these species in biological samples. Hence, a method was developed to exploit pH-dependent differences in the generation of arsines from inorganic and methylated arsenicals that contain either As(V) or As(III). Reduction with borohydride at pH 6 generated arsines from inorganic As(III), methyl As(III), and dimethyl As(III), but not from inorganic As(V), methyl As(V), and dimethyl As(V). Reduction with borohydride at pH 2 or lower generated arsines from arsenicals that contained either As(V) or As(III). Arsines are trapped in a liquid nitrogen-cooled gas chromatographic trap, which is subsequently warmed to allow separation of the hydrides by their boiling points. Atomic absorption spectrophotometry is used to detect and quantify the arsines. The detection limits (ng As ml(-1)) for inorganic As(III), methyl As(III), and dimethyl As(III) are 1.1, 1.2, and 6.5, respectively. This method has been applied to the analysis of arsenicals in water, human urine, and cultured cells. Both methyl As(III) and dimethyl As(III) are detected in urine samples from individuals who chronically consumed inorganic As-contaminated water and in human cells exposed in vitro to inorganic As(III). The reliable quantitation of inorganic and methylated arsenicals that contain As(III) in biological samples will aid the study of the toxicity of these species and may provide a new biomarker of the effects of chronic exposure to As.

Lead exposure in children living in a smelter community in region Lagunera, Mexico.

Industrial growth has created the potential for environmental problems in Mexico, since attention to environmental controls and urban planning has lagged behind the pace of industrialization. The aim of this cross-sectional study was to assess lead exposure in children aged 6-9 yr attending 3 primary schools and living in the vicinity of the largest smelter complex in Mexico. One of the schools is located 650 m distant from a smelter complex that includes a lead smelter (close school); the second is located 1750 m away from the complex and at the side of a heavy traffic road (intermediate school) in Torreon, Coahuila. The third school is located in Comez Palacio, Durango, 8100 m away from the smelter complex and distant from heavy vehicular traffic or industrial areas (remote school). Lead was measured in air, soil, dust, and well water. Lead in blood (PbB) was determined in 394 children attending the above mentioned schools. Determinations were performed by atomic absorption spectrometry. Diet, socioeconomic status, hygienic habits, and other variables were assessed by questionnaire. Median (range) PbB values were 7.8 microg/dl (3.54-29.61) in the remote school, 21.8 microg/dl (8.37-52.08) in the intermediate school and 27.6 microg/dl (7.37-58.53) in children attending the close school. The percentage of children with PbB > 15 microg/dl was 6.80%, 84.9%, and 92.1% respectively. In this order, the geometric means (range) of Pb concentrations in air were 2.5 microg/m3 (1.1-7.5), 5.8 microg/m3 (4.3-8.5), and 6.1 microg/m3 (1.6-14.9). The Pb concentrations in dust from playgrounds areas in the intermediate and close school settings ranged from 1,457 to 4,162.5 mg/kg. Pb concentrations in drinking water were less than 5 microg/L. Soil and dust ingestion and inhalation appear to be the main routes of exposure. Our results indicate that environmental contamination has resulted in an increased body burden of Pb, suggesting that children living in the vicinity of the smelter complex are at high risk for adverse effects of lead.

Arsenicals inhibit thioredoxin reductase in cultured rat hepatocytes.

Thioredoxin reductase (TR), an NADPH-dependent flavoenzyme that catalyzes the reduction of many disulfide-containing substrates, plays an important role in the cellular response to oxidative stress. Trivalent arsenicals, especially methyl As that contains trivalent arsenic (MAs(III)), are potent noncompetitive inhibitors of TR purified from mouse liver. Because MAs(III) is produced in the biomethylation of As, it was postulated that the extent of inhibition of TR in cultured rat hepatocytes would correlate with the intracellular concentration of methyl As. Exposure of cultured hepatocytes to inorganic As(III) (iAs(III)), MAs(III), or aurothioglucose (ATG, a competitive inhibitor of TR activity) for 30 min caused a concentration-dependent reduction in TR activity. The estimated IC(50) was >>100 microM for iAs(III), approximately 10 microM for ATG, and approximately 3 microM for MAs(III). In hepatocytes exposed to 1 microM MAs(III) for up to 24 h, the inhibition of TR activity was maximal ( approximately 40%) after exposure for 15 min. After exposure for 3 h [when most MAs(III) has been converted to dimethyl As (DMAs)], TR activity in these cells had returned to control levels. Notably, exposure of the cell to 50 microM DMAs(III) did not affect TR activity. In hepatocytes exposed to 10 microM iAs(III) for up to 24 h, the inhibition of TR activity was progressive; at 24 h, activity was reduced approximately 35%. Following exposure to iAs(III) or MAs(III), the extent of inhibition of TR activity correlated strongly with the intracellular concentration of MAs. Taken together, these results suggest that arsenicals formed in the course of cellular metabolism of As are potent inhibitors of TR activity. In particular, MAs(III), an intermediate in the metabolic pathway, is an especially potent inhibitor of TR. Hence, the capacity of cells to produce or consume the intermediates in the pathway for As methylation may be an important determinant of susceptibility to the toxic effects of As.

Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells.

Biomethylation is considered a major detoxification pathway for inorganic arsenicals (iAs). According to the postulated metabolic scheme, the methylation of iAs yields methylated metabolites in which arsenic is present in both pentavalent and trivalent forms. Pentavalent mono- and dimethylated arsenicals are less acutely toxic than iAs. However, little is known about the toxicity of trivalent methylated species. In the work reported here the toxicities of iAs and trivalent and pentavalent methylated arsenicals were examined in cultured human cells derived from tissues that are considered a major site for iAs methylation (liver) or targets for carcinogenic effects associated with exposure to iAs (skin, urinary bladder, and lung). To characterize the role of methylation in the protection against toxicity of arsenicals, the capacities of cells to produce methylated metabolites were also examined. In addition to human cells, primary rat hepatocytes were used as methylating controls. Among the arsenicals examined, trivalent monomethylated species were the most cytotoxic in all cell types. Trivalent dimethylated arsenicals were at least as cytotoxic as trivalent iAs (arsenite) for most cell types. Pentavalent arsenicals were significantly less cytotoxic than their trivalent analogs. Among the cell types examined, primary rat hepatocytes exhibited the greatest methylation capacity for iAs followed by primary human hepatocytes, epidermal keratinocytes, and bronchial epithelial cells. Cells derived from human bladder did not methylate iAs. There was no apparent correlation between susceptibility of cells to arsenic toxicity and their capacity to methylate iAs. These results suggest that (1) trivalent methylated arsenicals, intermediary products of arsenic methylation, may significantly contribute to the adverse effects associated with exposure to iAs, and (2) high methylation capacity does not protect cells from the acute toxicity of trivalent arsenicals.