Selenium modifies the metabolism and toxicity of arsenic in primary rat hepatocytes

Arsenic and selenium are metalloids with similar chemical properties and metabolic fates. Inorganic arsenic (iAs) has been shown to modify metabolism and toxicity of inorganic and organic selenium compounds. However, little is known about effects of selenium on metabolism and toxicity of iAs. The present work examines the effects of selenite (Se(IV)) on the cellular retention, methylation, and cytotoxicity of trivalent iAs, arsenite (iAs(III)), in primary cultures of rat hepatocytes. The concurrent exposure to Se(IV) (0.1 to 6 microM) inhibited methylation and/or significantly increased cellular retention of iAs(III) in cultured cells. The ratio of the methylated metabolites produced from iAs(III), dimethylarsenic (DMAs) to methylarsenic (MAs), decreased considerably in cells treated with Se(IV), suggesting that synthesis of DMAs from MAs may be more susceptible to inhibition by Se(IV) than is the production of MAs from iAs(III). The 24-h preexposure to 2 microM Se(IV) had a similar but less pronounced inhibitory effect on the methylation of iAs(III) in cultured cells. The exposure to 2 microM Se(IV) alone for up to 24 h had no effect on the viability of cultured hepatocytes. However, concurrent exposure to 2 microM Se(IV) increased the cytotoxicity of iAs(III) and its mono- and dimethylated metabolites that contain trivalent arsenic, MAs(III) and DMAs(III). These data suggest that pre- or coexposure to inorganic selenium may enhance the toxic effects of iAs, increasing its retention in tissues and suppressing its methylation, which may be a pathway for detoxification of iAs.     

Arsenite uptake and metabolism by rat hepatocyte primary cultures in comparison with kidney- and hepatocyte-derived rat cell lines.

Biotransformation by methylation to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) influences inorganic arsenical toxicity, which is often investigated in cultured cells. Arsenic (III) uptake and methylation was assessed in rat hepatocytes in primary culture and in three established rat cell lines (hepatoma-derived McA-RH 7777 cells and H4-II-EC-3 cells, and kidney epithelium-derived NRK-52E cells) to compare their use as model systems for arsenite metabolism. Incubation of all cell types with 0.27, 0.67, 1.33, 2.67, or 6.67 microM As(III) concentrations resulted in concentration-dependent arsenic uptake and biomethylation. Arsenic uptake by the NRK-52E cells was initially slower than that of the other cells, but by 8 h, total uptake was similar in all cell types. At the lowest arsenite concentration, the percentages of total arsenic methylated to MMA and DMA by the hepatocytes and the McA-RH 7777 cells were similar (67 and 66%); methylation by the H4-II-EC-3 cells was somewhat lower (52%), and methylation by the kidney-derived NRK-52E cells was much lower (15%). Total arsenic methylation was inhibited in the cell lines, but not in the hepatocytes, at the highest arsenite concentrations. In all cases, exposure to increased arsenite concentrations inhibited conversion of MMA to DMA much more than it affected the initial methylation step (inorganic arsenite to MMA). These results indicate that rat hepatocytes in primary culture and established rat hepatoma-derived cell lines are similar in their abilities to accumulate and methylate arsenic to MMA and DMA at environmentally relevant arsenic concentrations in the medium. They differed from the kidney epithelium-derived cells, which exhibited substantially lower biomethylation activity.     

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.     

Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes

Liver is a prime site for conversion of inorganic arsenic (iAs) to methylated metabolites, including methylarsenicals (MAs) and dimethylarsenicals (DMAs). To assess interindividual variation in the capacity of liver to metabolize iAs, we examined the metabolic fate of arsenite (iAs(III)) in normal primary human hepatocytes obtained from eight donors and cultured under standard conditions. Methylation rates, yields, and distribution of arsenicals were determined for hepatocytes exposed to 0.3-30 nmol of iAs(III)/mg of protein for 24 h. Although the accumulation of arsenic (As) by cells was a linear function of the initial concentration of iAs(III) in culture, the concentration of As retained in cells varied several fold among donors. DMAs was the major methylated metabolite found in cultures exposed to low concentrations of iAs(III); at higher concentrations, MAs was always predominant. Maximal rates for methylation of iAs(III) were usually attained at 3 or 9 nmol of iAs(III)/mg of protein and varied about 7-fold among donors. For most donors, the methylation rate decreased at the highest iAs(III) concentrations. MAs was the major methylated metabolite retained in cells regardless of exposure level. DMAs was the major methylated metabolite found in medium. The interindividual differences in rates for iAs(III) methylation were not strictly associated with variations in basal mRNA levels for cyt19, an As-methyltransferase. Analysis of the coding sequence of cyt19 identified one heterozygote with Met287Thr mutation in a single allele. Thus, genetic polymorphism of cyt19 along with other cellular factors is likely responsible for interindividual differences in the capacity of primary human hepatocytes to retain and metabolize iAs(III).     

Metabolism of arsenic in primary cultures of human and rat hepatocytes.

The liver is considered a major site for methylation of inorganic arsenic (iAs). However, there is little data on the capacity of human liver to methylate iAs. This work examined the metabolism of arsenite (iAs(III)), arsenate (iAs(V)), methylarsine oxide (MAs(III)O), methylarsonic acid (MAs(V)), dimethylarsinous acid (DMAs(III)), and dimethylarsinic acid (DMAs(V)) in primary cultures of normal human hepatocytes. Primary rat hepatocytes were used as methylating controls. iAs(III) and MAs(III)O were metabolized more extensively than iAs(V) and MAs(V) by either cell type. Neither human nor rat hepatocytes metabolized DMAs(III) or DMAs(V). Methylation of iAs(III) by human hepatocytes yielded methylarsenic (MAs) and dimethylarsenic (DMAs) species; MAs(III)O was converted to DMAs. The total methylation yield (MAs and DMAs) increased over the range of 0.1 to 4 microM iAs(III). However, DMAs production was inhibited by iAs(III) in a concentration-dependent manner, and the DMAs/MAs ratio decreased. iAs(III) (10 and 20 microM) inhibited both methylation reactions. Inhibition of DMAs synthesis resulted in accumulation of iAs and MAs in human hepatocytes, suggesting that dimethylation is required for iAs clearance from cells. Methylation capacities of human hepatocytes obtained from four donors ranged from 3.1 to 35.7 pmol of iAs(III) per 10(6) cells per hour and were substantially lower than the methylation capacity of rat hepatocytes (387 pmol of iAs(III) per 10(6) cells per hour). The maximal methylation rates for either rat or human hepatocytes were attained between 0.4 and 4 microM iAs(III). In summary, (i) human hepatocytes methylate iAs, (ii) the capacities for iAs methylation vary among individuals and are saturable, and (iii) moderate concentrations of iAs inhibit DMAs synthesis, resulting in an accumulation of iAs and MAs in cells.     

Effect of methylated forms of selenium on cell viability and the induction of DNA strand breakage.

Selenobetaine (SB) and selenobetaine methyl ester (SBME) are methylated selenonium derivatives that undergo metabolism to release methyl selenide and dimethylselenide, respectively, as primary metabolites. Since methylation of selenium is considered to be detoxifying, the toxicologic activity of SB or SBME may differ from that of inorganic forms of selenium, such as selenite, that undergo reduction and can induce cell damage. In this study, the effects of SB, SBME and selenite on the viability and long-term growth potential of a mouse leukemia cell line (L1210) were compared. Treatment with 20 microM selenite reduced the rate of cell doubling and the long-term growth potential of cells as measured by colony-forming ability. These effects of selenite were accompanied by a reduction in DNA integrity, assessed by alkaline elution analysis for single-strand breaks. Exposure to 500 microM SB or SBME for 24 hr reduced the colony-forming ability of cells in the absence of any effect on dye exclusion or induction of single-strand breaks in DNA. Exposure of cells to 500 microM SB or SBME resulted in levels of intracellular selenium similar to those after exposure to 20 microM selenite. These observations indicate that it is possible to maintain high intracellular levels of selenium, by exposure to methylated selenocompounds, without affecting DNA integrity. These findings also suggest that DNA fragmentation resulting from exposure to selenite occurs during its reductive metabolism and not from the accumulation of a methylated metabolite of selenium. The fact that SB or SBME reduced the ability of L1210 cells to form colonies in agar in the absence of either DNA fragmentation or any effect on the ability of treated cells to exclude a vital dye suggests that both methylated compounds alter the long-term proliferative potential of cells via a mechanism(s) distinct from that associated with cell injury and death by necrosis. Efforts are underway to determine the origin of these effects.     

Monomethylarsonous acid (MMA(III)) is more toxic than arsenite in Chang human hepatocytes

Methylation has been considered to be the primary detoxication pathway of inorganic arsenic. Inorganic arsenic is methylated by many, but not all animal species, to monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), and dimethylarsinic acid (DMA(V)). The As(V) derivatives have been assumed to produce low toxicity, but the relative toxicity of MMA(III) remains unknown. In vitro toxicities of arsenate, arsenite, MMA(V), MMA(III), and DMA(V) were determined in Chang human hepatocytes. Leakage of lactate dehydrogenase (LDH) and intracellular potassium (K(+)) and mitochondrial metabolism of the tetrazolium salt XTT were used to assess cytotoxicity due to arsenic exposure. The mean LC50 based on LDH assays in phosphate media was 6 microM for MMA(III) and 68 microM for arsenite. Using the assay for K(+) leakage in phosphate media, the mean LC50 was 6.3 microM for MMA(III) and 19.8 microM for arsenite. The mean LC50 based on the XTT assay in phosphate media was 13.6 microM for MMA(III) and 164 microM for arsenite. The results of the three cytotoxicity assays (LDH, K(+), and XTT) reveal the following order of toxicity in Chang human hepatocytes: MMA(III) > arsenite > arsenate > MMA(V) = DMA(V). Data demonstrate that MMA(III), an intermediate in inorganic arsenic methylation, is highly toxic and again raises the question as to whether methylation of inorganic arsenic is a detoxication process.     

Metabolism and toxicity of arsenic in human urothelial cells expressing rat arsenic (+3 oxidation state)-methyltransferase

The enzymatic methylation of inorganic As (iAs) is catalyzed by As(+3 oxidation state)-methyltransferase (AS3MT). AS3MT is expressed in rat liver and in human hepatocytes. However, AS3MT is not expressed in UROtsa, human urothelial cells that do not methylate iAs. Thus, UROtsa cells are an ideal null background in which the role of iAs methylation in modulation of toxic and cancer-promoting effects of this metalloid can be examined. A retroviral gene delivery system was used in this study to create a clonal UROtsa cell line (UROtsa/F35) that expresses rat AS3MT. Here, we characterize the metabolism and cytotoxicity of arsenite (iAs(III)) and methylated trivalent arsenicals in parental cells and clonal cells expressing AS3MT. In contrast to parental cells, UROtsa/F35 cells effectively methylated iAs(III), yielding methylarsenic (MAs) and dimethylarsenic (DMAs) containing either As(III) or As(V). When exposed to MAs(III), UROtsa/F35 cells produced DMAs(III) and DMAs(V). MAs(III) and DMAs(III) were more cytotoxic than iAs(III) in UROtsa and UROtsa/F35 cells. The greater cytotoxicity of MAs(III) or DMAs(III) than of iAs(III) was associated with greater cellular uptake and retention of each methylated trivalent arsenical. Notably, UROtsa/F35 cells were more sensitive than parental cells to the cytotoxic effects of iAs(III) but were more resistant to cytotoxicity of MAs(III). The increased sensitivity of UROtsa/F35 cells to iAs(III) was associated with inhibition of DMAs production and intracellular accumulation of MAs. The resistance of UROtsa/F35 cells to moderate concentrations of MAs(III) was linked to its rapid conversion to DMAs and efflux of DMAs. However, concentrations of MAs(III) that inhibited DMAs production by UROtsa/F35 cells were equally toxic for parental and clonal cell lines. Thus, the production and accumulation of MAs(III) is a key factor contributing to the toxicity of acute iAs exposures in methylating cells.     

Interspecies differences in metabolism of arsenic by cultured primary hepatocytes

Biomethylation is the major pathway for the metabolism of inorganic arsenic (iAs) in many mammalian species, including the human. However, significant interspecies differences have been reported in the rate of in vivo metabolism of iAs and in yields of iAs metabolites found in urine. Liver is considered the primary site for the methylation of iAs and arsenic (+3 oxidation state) methyltransferase (As3mt) is the key enzyme in this pathway. Thus, the As3mt-catalyzed methylation of iAs in the liver determines in part the rate and the pattern of iAs metabolism in various species. We examined kinetics and concentration-response patterns for iAs methylation by cultured primary hepatocytes derived from human, rat, mice, dog, rabbit, and rhesus monkey. Hepatocytes were exposed to [⁷³As]arsenite (iAs^III; 0.3, 0.9, 3.0, 9.0 or 30 nmol As/mg protein) for 24 h and radiolabeled metabolites were analyzed in cells and culture media. Hepatocytes from all six species methylated iAs^III to methylarsenic (MAs) and dimethylarsenic (DMAs). Notably, dog, rat and monkey hepatocytes were considerably more efficient methylators of iAs^III than mouse, rabbit or human hepatocytes. The low efficiency of mouse, rabbit and human hepatocytes to methylate iAs^III was associated with inhibition of DMAs production by moderate concentrations of iAs^III and with retention of iAs and MAs in cells. No significant correlations were found between the rate of iAs methylation and the thioredoxin reductase activity or glutathione concentration, two factors that modulate the activity of recombinant As3mt. No associations between the rates of iAs methylation and As3mt protein structures were found for the six species examined. Immunoblot analyses indicate that the superior arsenic methylation capacities of dog, rat and monkey hepatocytes examined in this study may be associated with a higher As3mt expression. However, factors other than As3mt expression may also contribute to the interspecies differences in the hepatocyte capacity to methylate iAs.     

Monomethylarsonous acid (MMA(III)) and arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate dehydrogenase.

Monomethylarsonous acid (MMA(III)), a metabolite of inorganic arsenic, has received very little attention from investigators of arsenic metabolism in humans. MMA(III), like sodium arsenite, contains arsenic in the +3 oxidation state. Although we have previously demonstrated that it is more toxic than arsenite in cultured Chang human hepatocytes, there are no data showing in vivo toxicity of MMA(III). When MMA(III) or sodium arsenite was administered intraperitoneally to hamsters, the LD(50)s were 29.3 and 112.0 micromol/kg of body wt, respectively. In addition, inhibition of hamster kidney or purified porcine heart pyruvate dehydrogenase (PDH) activity by MMA(III) or arsenite was determined. To inhibit hamster kidney PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide, MMA(III) as diiodomethylarsine, and arsenite were 59.9 +/- 6.5, 62.0 +/- 1.8, and 115.7 +/- 2.3 microM, respectively. To inhibit activity of purified porcine heart PDH activity by 50%, the concentrations (mean +/- SE) of MMA(III) as methylarsine oxide and arsenite were 17.6 +/- 4.1 and 106.1 +/- 19.8 microM, respectively. These data demonstrate that MMA(III) is more toxic than inorganic arsenite, both in vivo and in vitro, and call into question the hypothesis that methylation of inorganic arsenic is a detoxication process.     

Effect of in vitro treatment of rat hepatocytes with selenium, and/or cadmium on cell viability, glucose output, and cellular glutathione

The effects of cadmium as cadmium acetate and selenium as sodium selenite on glucose output, cell viability, and glutathione levels in rat hepatocytes were evaluated. Isolated hepatocytes (200 mg wet wt cells) derived from naive male Sprague--Dawley rats (210-260 g) were incubated at 37 degrees C, with sodium acetate (C2H3NaO2; NaAc) 12.5 microM, 6.3 microM, 3.2 microM; cadmium acetate (C4H6CdO4; Cd) 12.5 microM, 6.3 microM, 3.2 microM; sodium selenite (Na2SeO3; Se) 25 microM, 12.5 microM, 6.3 microM; or Se (6.3 microM) and Cd (3.2 microM). After an incubation period of 2 h, glucose output, cell viability, and reduced glutathione (GSH) levels were determined. The results obtained indicate that incubation of hepatocytes with Se (12.5 or 25 microM) or Cd (3.2, 6.3, or 12.5 microM) resulted in a significant decrease in glucose output, cell viability, and glutathione levels (P less than 0.05) when compared to those incubated with NaAc (control). Selenium in concentrations of 6.3 microM decreased glutathione levels and cell viability only. The damaging effects induced by Cd on hepatocytes were significantly greater than those induced by Se. The decrease in glutathione levels observed following Cd treatment was considerably lowered when Se was concurrently added to the incubation medium. These findings suggest that Se may in part protect against the deleterious effects of Cd on hepatocytes.     

Release of apoptotic cytochrome C from mitochondria by dimethylarsinous acid occurs through interaction with voltage-dependent anion channel in vitro

Arsenic is known to be a human carcinogen as well as one of the most effective drugs for treatment of patients with acute promyelocytic leukemia. The intermediate metabolites of arsenic, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), are formed by methylation reactions, and they are more reactive and toxic than the inorganic precursor arsenite (iAs(III)); however, the detailed mechanism of toxicity is poorly understood. Here, we studied the effects of three arsenic compounds (i.e., iAs(III), MMA(III), and DMA(III)) on mitochondrial permeability transition pore (mPTP) and release of apoptotic cytochrome c (Cyt c) after incubating with rat liver mitochondria. Inorganic iAs(III) had no effect on mitochondrial swelling even at higher concentrations ranging up to 100 μM, but swelling was significantly induced in the presence of Ca(2+). Additionally, mitochondrial swelling was strongly induced by exposure to the methylated forms of MMA(III) and DMA(III) in a dose-dependent manner in the absence of Ca(2+), suggesting that the methylated forms may have potent effects on cellular mitochondria. Although mitochondrial swelling was completely inhibited in the presence of cyclosporin-A (an inhibitor of mitochondrial permeability transition) or ruthenium red (an inhibitor of Ca(2+) uniporter) following exposure to methylated arsenicals, the release of apoptotic Cyt c from mitochondria was not inhibited, indicating that release of Cyt c is probably not dependent on mPTP opening. In addition, inhibitors of Bax (e.g., Bax-inhibiting peptide) did not reduce the release of Cyt c from the mitochondria by formation of Bax-voltage-dependent anion channel (VDAC) complex, whereas the recombinant Bcl-x(L )proteins significantly reduced the release of Cyt c after exposure to DMA(III), suggesting that dimethylated DMA(III) directly interacted with the VDAC in mitochondria and caused the release of Cyt c from mitochondria.     

Effects of ascorbic acid on selenium teratogenicity in cultured rat embryos

The effects of ascorbic acid, as an exogenous antioxidant, on selenium (Se) teratogenicity were examined using rat embryo culture. Rat embryos at day 9.5 of gestation were cultured for 48 h in the presence of sodium selenite at 10 and 20 microM or sodium selenate at 30 and 100 microM with or without the addition of 1 mM of L-ascorbic acid (AsA). Selenite or selenate alone increased the incidence of embryonic malformation. With AsA, the incidence of selenite-induced embryonic malformation was increased. On the contrary, the incidence of selenate-induced embryonic malformation was decreased with AsA. It was considered from these results that the redox states in the embryonic environment and of Se are critical in Se teratogenicity.     

Metabolism and toxicity of benzophenone in isolated rat hepatocytes and estrogenic activity of its metabolites in MCF-7 cells

The metabolism and cytotoxicity of benzophenone and estrogenic activity of its metabolites have been studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with benzophenone (0.25-1.0 mM) elicited a concentration- and time-dependent cell death, accompanied by loss of intracellular ATP and depletion of adenine nucleotide pools. Benzophenone at a low-toxic level (0.25 mM) in the hepatocyte suspensions was converted to benzhydrol, p-hydroxybenzophenone and its sulfate conjugate, without marked loss of cell viability. The amounts of benzhydrol and sulfate conjugate increased with time. In contrast, addition of 2,6-dichloro-4-nitrophenol (an inhibitor of sulfotransferase; 0.1 mM), nontoxic to hepatocytes during the incubation period, enhanced benzophenone-induced cytotoxicity, and this effect was accompanied by a decrease in the formation of sulfate conjugate and increase in the amount of free p-hydroxybenzophenone. In another experiment, MCF-7 cells, estrogen-responsible breast cancer cells were cultured in estradiol free medium and then exposed to 10 nM-500 microM benzophenone or its metabolites for 6 days. Although at higher concentrations all the compounds were toxic, except for benzophenone and benzhydrol, 10-100 microM p-hydroxybenzophenone significantly increased cell proliferation. These results indicate that benzophenone is enzymaticaly converted to benzhydrol, p-hydroxybenzophenone and its sulphate conjugate in rat hepatocytes. Even if there is less free p-hydroxybenzophenone than benzhydrol and sulfate conjugate in hepatocyte suspensions, p-hydroxybenzophenone itself acts as a weak xeno-estrogen on MCF-7 cells.     

Glutathione modulates recombinant rat arsenic (+3 oxidation state) methyltransferase-catalyzed formation of trimethylarsine oxide and trimethylarsine

Humans and other species enzymatically convert inorganic arsenic (iAs) into methylated metabolites. Although the major metabolites are mono- and dimethylated arsenicals, trimethylated arsenicals have been detected in urine following exposure to iAs. The AS3MT gene encodes an arsenic (+3 oxidation state) methyltransferase, which catalyzes both the oxidative methylation of trivalent arsenicals and the reduction of pentavalent arsenicals. In reaction mixtures containing recombinant rat AS3MT (rrAS3MT) and radiolabeled arsenite, mono- and dimethylated arsenicals and a third radiolabeled product can be resolved by thin-layer chromatography. Hydride generation atomic absorption spectrometry and electrospray ionization mass spectrometry identified the third reaction product as trimethylarsine oxide. The addition of glutathione to reaction mixtures containing radiolabeled arsenite and rrAS3MT increased the yield of methylated and dimethylated arsenicals but suppressed the formation of trimethylarsine oxide. Although a dimethylarsenic-glutathione complex was rapidly converted to trimethylarsine oxide, the addition of a molar excess of glutathione to dimethylarsenic suppressed the production of trimethylarsine oxide. The nonquantitative recovery of radioarsenic from reaction mixtures suggested that AS3MT catalyzed the formation of a volatile arsenical. This volatile species was identified as trimethylarsine. Thus, AS3MT catalyzes the formation of all products in a reaction sequence leading from an inorganic to a volatile methylated arsenical. The regulation of this pathway by intracellular glutathione may be an important determinant of the pattern and extent of formation of arsenicals.     

Interactions between selenium and group Va-metalloids (arsenic, antimony and bismuth) in the biliary excretion

The interrelationship between the biliary excretion of exogenous group Va-metalloids (arsenic, antimony and bismuth) and selenium, as well as endogenous glutathione has been studied in rats injected intravenously with sodium selenite and one of the group Va-metalloids. Arsenic, antimony and bismuth appeared in the bile of rats together with large amounts of non-protein thiols (NPSH, representing glutathione and its SH-containing degradation products) and, with the exception of bismuth, they caused choleresis. Significant interactions were observed in the hepatobiliary disposition between selenium and each of the group Va-metalloids, however, their outcomes were not uniform. When coadministered with sodium arsenite or arsenate, selenite enhanced the initial biliary excretion of arsenic 2- and 8-fold, respectively, without further increasing the concomitant excretion of NPSH or the choleretic effect of arsenicals. However, selenite augmented neither the excretion of antimony or bismuth, nor the simultaneous biliary release of NPSH. In turn, arsenite, arsenate and antimony potassium tartrate increased the initial biliary excretion of selenium more than 10-fold and enhanced the accumulation of selenium in blood (exclusively in the erythrocytes). In contrast, administration of bismuth ammonium citrate diminished both the biliary excretion and the erythrocytic accumulation of selenium, while causing retention of selenium in the blood plasma. In rats receiving arsenic or antimony with selenite, the time courses of the biliary excretion of these group Va-metalloids, selenium and NPSH were similar. It is hypothesised that incorporation of selenol metabolites of selenite into the glutathione complexes of arsenic and antimony, resulting in cholephilic ternary complexes, accounts for the arsenic- and antimony-induced augmentation of the hepatobiliary transport of selenium. However, additional chemical and/or dispositional mechanisms are thought to be responsible for the selenite-induced increase in biliary excretion of arsenic.     

Effect of arsenite on induction of CYP1A, CYP2B, and CYP3A in primary cultures of rat hepatocytes.

In earlier studies, sodium arsenite treatment was shown to decrease induction of enzymatic activities associated with hepatic CYPs in rats. Here we investigated the effect of sodium arsenite on induction of CYP2B, CYP1A, and CYP3A in primary cultures of rat hepatocytes. Arsenite decreased the induction of all three families of CYP, as measured enzymatically and immunochemically. These decreases in CYPs occurred at concentrations of arsenite (2.5-10 microM) at which no toxicity was observed; however, toxicity was observed at 25 microM arsenite. With 3-methylcholanthrene as inducer, 5 microM arsenite caused a 55% decrease in CYP1A1 immunoreactive protein and enzyme activity, but only a 25% decrease in CYP1A1 mRNA. With phenobarbital (PB) as the inducer, 2.5 microM arsenite decreased CYP2B enzyme activity and immunoreactive protein 50%, with only a 25% decrease in CYP2B1 mRNA. 5 microM Arsenite decreased CYP2B enzyme activity and immunoreactive protein 80%, but decreased CYP2B1 mRNA only 50%, while CYP3A protein was decreased greater than 75% with no decrease in CYP3A23 mRNA. With dexamethasone (DEX) as inducer, 5 microM sodium arsenite caused a 50% decrease in immunoreactive CYP3A and a 30% decrease in CYP3A23 mRNA. Although arsenite-mediated increases in heme oxygenase (HO) inversely correlated with decreases in CYP2B or CYP1A activity, inclusion of heme in cultures treated with inducers of CYP1A or CYP2B did not prevent the arsenite-mediated decreases in these CYPs. Even though added heme induced HO to similar levels with and without arsenite, decreases in CYPs were only observed in the presence of arsenite. These results suggest that, in rat hepatocytes, elevated levels of HO alone are not responsible for arsenite-mediated decreases in CYP.     

Hepatoprotective and hepatotoxic activities of sophoradiol analogs on rat primary liver cell cultures

As a part of our studies of hepatoprotective drugs, we prepared kaikasaponin I (2), sophoradiol monoglucuronide (SoMG, 3) and sophoradiol (4) from kaikasaponin III (1). We examined the hepatoprotective effects of these analogs, using immunologically-induced liver injury in primary cultured rat hepatocytes and found that compound 1 was more effective than soyasaponin I (1a) while 2 was more effective than 1. On the other hand, 3 was less effective than 2 at 30-200 microm. Further, compound 3 was strongly cytotoxic at 500 microM while 4 exhibited hepatoprotective activity at the same dose, although less potent. When the cytotoxicity toward hepatocytes of these analogs was tested, only 3 was cytotoxic at doses of 200 and 500 microM. This is the first example of an oleanene glucuronide (OG) which is cytotoxic toward hepatocytes. Compound 3 exhibited hepatoprotective activity at 200 microM, while it was also cytotoxic at the same dose without antiserum. Therefore, the hepatoprotective activity of OG represents a balance between a hepatoprotective action and its cytotoxicity toward hepatocytes.     

Further evidence against a direct genotoxic mode of action for arsenic-induced cancer

Arsenic in drinking water, a mixture of arsenite and arsenate, is associated with increased skin and other cancers in Asia and Latin America, but not the United States. Arsenite alone in drinking water does not cause skin cancers in experimental animals; therefore, it is not a complete carcinogen in skin. We recently showed that low concentrations of arsenite enhanced the tumorigenicity of solar UV irradiation in hairless mice, suggesting arsenic cocarcinogenesis with sunlight in skin cancer and perhaps with different carcinogenic partners for lung and bladder tumors. Cocarcinogenic mechanisms could include blocking DNA repair, stimulating angiogenesis, altering DNA methylation patterns, dysregulating cell cycle control, induction of aneuploidy and blocking apoptosis. Arsenicals are documented clastogens but not strong mutagens, with weak mutagenic activity reported at highly toxic concentrations of inorganic arsenic. Previously, we showed that arsenite, but not monomethylarsonous acid (MMA[III]), induced delayed mutagenesis in HOS cells. Here, we report new data on the mutagenicity of the trivalent methylated arsenic metabolites MMA(III) and dimethylarsinous acid [DMA(III)] at the gpt locus in Chinese hamster G12 cells. Both methylated arsenicals seemed mutagenic with apparent sublinear dose responses. However, significant mutagenesis occurred only at highly toxic concentrations of MMA(III). Most mutants induced by MMA(III) and DMA(III) exhibited transgene deletions. Some non-deletion mutants exhibited altered DNA methylation. A critical discussion of cell survival leads us to conclude that clastogenesis occurs primarily at highly cytotoxic arsenic concentrations, casting further doubt as to whether a genotoxic mode of action (MOA) for arsenicals is supportable.