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.     

Effects of folate on arsenic toxicity in Chang human hepatocytes: Involvement of folate antioxidant properties

We investigated the effects and modes of action of the nutritional factor folate on arsenic-induced toxicity in Chang human hepatocytes. Cells were cultured in folate-deficient medium, normal folate medium or folate-supplemented medium for 1 h and then co-treated with or without 20-μM sodium arsenite (NaAsO₂) for 24 h. The results showed that folate deficiency significantly aggravated the NaAsO₂-induced apoptotic progression [evidenced by phosphatidylserine externalization, cleavage of capspase-3 and poly (ADP-ribose) polymerase (PARP), collapse of mitochondrial potential, and release of cytochrome c from the mitochondria] and decrease of cell viability. Folate supplementation significantly attenuated all the above mentioned NaAsO₂-induced effects except phosphatidylserine externalization. The NaAsO₂-induced generation of intracellular reactive oxygen species and malondialdehyde was aggravated, to some extent, by folate deficiency, but these phenomena were significantly suppressed by folate supplementation. In contrast, NaAsO₂-induced elevation of reduced glutathione levels was significantly suppressed by folate deficiency, but significantly enhanced by folate supplementation. In addition, folate deficiency significantly decreased the arsenic methylation capacity of the hepatocytes, but had no effects on cellular retention of arsenic. Folate supplementation had no significant effect on cellular retention or methylation of arsenic. These results indicate that folate deficiency aggravates arsenic-induced toxicity and apoptosis, while folate supplementation attenuates these effects. Folate, which plays a role in arsenic metabolism, also exerts its effect on arsenic toxicity at least partly because of its antioxidant property.     

Molecular processes in cellular arsenic metabolism

Elucidating molecular processes that underlie accumulation, metabolism and binding of iAs and its methylated metabolites provides a basis for understanding the modes of action by which iAs acts as a toxin and a carcinogen. One approach to this problem is to construct a conceptual model that incorporates available information on molecular processes involved in the influx, metabolism, binding and efflux of arsenicals in cells. This conceptual model is initially conceived as a non-quantitative representation of critical molecular processes that can be used as a framework for experimental design and prediction. However, with refinement and incorporation of additional data, the conceptual model can be expressed in mathematical terms and should be useful for quantitative estimates of the kinetic and dynamic behavior of iAs and its methylated metabolites in cells. Development of a quantitative model will be facilitated by the availability of tools and techniques to manipulate molecular processes underlying transport of arsenicals across cell membranes or expression and activity of enzymes involved in methylation of arsenicals. This model of cellular metabolism might be integrated into more complex pharmacokinetic models for systemic metabolism of iAs and its methylated metabolites. It may also be useful in development of biologically based dose-response models describing the toxic and carcinogenic actions of arsenicals.     

Effect of arsenic on carbohydrate metabolism after single or repeated injection in guinea pigs

Divergent pattern in pyruvate efflux from livers perfused with As₂O₃ and livers of animals previously repeatedly treated with the toxicant was observed in earlier experiments (Reichl et al. 1987, 1988). Further studies of the effect of As₂O₃ on carbohydrate metabolism were therefore performed. Male guinea pigs received either a single dose of As₂O₃ 10 mg· kg^–1 s.c. or repeated doses of 2.5 mg·kg^–1 bis in die (b.i.d.) on 5 consecutive days. One hour after the single dose or 1 h and 16 h after the last injection in the repeated treatment group the animals were sacrificed under anaesthesia. The livers were removed by a freeze-stop technique and the contents of glycogen and glycolysis intermediates were measured. In the single dose group a decrease in fructose-1,6-diphosphate and glycerolaldehyde-3-phosphate and an increase in phosphoenolpyruvate and pyruvate was observed. In the repeat dose, 1-h group a significant decrease in glycogen, glucose-6-phosphate, fructose-6-phosphate, glycerolaldehyde-3-phosphate, dihydroxyacetonephosphate, 2-phosphoglycerate and pyruvate was found. In the repeat dose, 16-h group the contents of glycogen, glucose-6-phosphate, pyruvate and lactate were diminished. The most prominent finding after repeated As₂O₃ administration was a marked depletion in total carbohydrate content. This was due mainly to depletion of glycogen.     

Individual differences in arsenic metabolism and lung cancer in a case-control study in Cordoba, Argentina

In humans, ingested inorganic arsenic is metabolized to monomethylarsenic (MMA) then to dimethylarsenic (DMA), although in most people this process is not complete. Previous studies have identified associations between the proportion of urinary MMA (%MMA) and increased risks of several arsenic-related diseases, although none of these reported on lung cancer. In this study, urinary arsenic metabolites were assessed in 45 lung cancer cases and 75 controls from arsenic-exposed areas in Cordoba, Argentina. Folate has also been linked to arsenic-disease susceptibility, thus an exploratory assessment of associations between single nucleotide polymorphisms in folate metabolizing genes, arsenic methylation, and lung cancer was also conducted. In analyses limited to subjects with metabolite concentrations above detection limits, the mean %MMA was higher in cases than in controls (17.5% versus 14.3%, p = 0.01). The lung cancer odds ratio for subjects with %MMA in the upper tertile compared to those in the lowest tertile was 3.09 (95% CI, 1.08-8.81). Although the study size was too small for a definitive conclusion, there was an indication that lung cancer risks might be highest in those with a high %MMA who also carried cystathionine β-synthase (CBS) rs234709 and rs4920037 variant alleles. This study is the first to report an association between individual differences in arsenic metabolism and lung cancer, a leading cause of arsenic-related mortality. These results add to the increasing body of evidence that variation in arsenic metabolism plays an important role in arsenic-disease susceptibility.     

Accumulation and metabolism of arsenic in mice after repeated oral administration of arsenate

Exposure to the human carcinogen inorganic arsenic (iAs) occurs daily. However, the disposition of arsenic after repeated exposure is not well known. This study examined the disposition of arsenic after repeated po administration of arsenate. Whole-body radioassay of adult female B6C3F1 mice was used to estimate the terminal elimination half-life of arsenic after a single po dose of [(73)As]arsenate (0.5 mg As/kg). From these data, it was estimated that steady-state levels of whole-body arsenic could be attained after nine repeated daily doses of [(73)As]arsenate (0.5 mg As/kg). The mice were whole-body radioassayed immediately before and after the repeated dosing. Excreta were collected daily and analyzed for arsenic-derived radioactivity and arsenicals. Whole-body radioactivity was determined 24 h after the last repeated dose, and five mice were then euthanized and tissues analyzed for radioactivity. The remaining mice were whole-body radioassayed for 8 more days, and then their tissues were analyzed for radioactivity. Other mice were administered either a single or nine repeated po doses of non-radioactive arsenate (0.5 mg As/kg). Twenty-four hours after the last dose, the mice were euthanized, and tissues were analyzed for arsenic by atomic absorption spectrometry (AAS). Whole-body radioactivity was rapidly eliminated from mice after repeated [(73)As]arsenate exposure, primarily by urinary excretion in the form of dimethylarsinic acid (DMA(V)). Accumulation of radioactivity was highest in bladder, kidney, and skin. Loss of radioactivity was most rapid in the lung and slowest in the skin. There was an organ-specific distribution of arsenic as determined by AAS. Monomethylarsonic acid was detected in all tissues except the bladder. Bladder and lung had the highest percentage of DMA(V) after a single exposure to arsenate, and it increased with repeated exposure. In kidney, iAs was predominant. There was a higher percentage of DMA(V) in the liver than the other arsenicals after a single exposure to arsenate. The percentage of hepatic DMA(V) decreased and that of iAs increased with repeated exposure. A trimethylated metabolite was also detected in the liver. Tissue accumulation of arsenic after repeated po exposure to arsenate in the mouse corresponds to the known human target organs for iAs-induced carcinogenicity.     

Functionality of cultured human hepatocytes from elective samples, cadaveric grafts and hepatectomies

The major possible sources of human liver for hepatocyte isolation are elective liver biopsies, cadaveric liver grafts and therapeutic liver resections. The suitability in terms of metabolic-competent hepatocyte cultures and risk/benefit of these resources has been comparatively studied. To this end, viability of isolated hepatocytes, yield of isolation procedure, hepatocyte survival during culture and CYP activities were the parameters analysed. The best results were found in hepatocytes prepared from elective biopsies, whereas a marked reduction in viability and functional competence was seen in hepatocytes from hepatectomy samples. Metabolic differences were observed in total CYP oxidative metabolism (7-ethoxycoumarin O-deethylation, total testosterone hydroxylation), as well as in CYP3A4, CYP2C9 or CYP2C19 activities (testosterone oxidations at 6β-, 16β- and 17-positions, respectively). Vascular control during the hepatectomy procedure influenced hepatocyte functionality: higher CYP activities were found in hepatocytes isolated from samples obtained under non-ischemic conditions or continuous vascular clamping than in those obtained under intermittent vascular clamping. In addition to cellular functionality, other criteria such as sample availability or ethical aspects should be considered. Elective biopsies have low, but not absent, surgical risk. However, the better functionality and the higher accessibility of elective liver samples in comparison to the other groups suggest this source of liver tissue as the most appropriate for cell harvesting purposes.     

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.     

Urinary arsenic metabolism in a Western Chinese population exposed to high-dose inorganic arsenic in drinking water: Influence of ethnicity and genetic polymorphisms

To investigate the differences in urinary arsenic metabolism patterns of individuals exposed to a high concentration of inorganic arsenic (iAs) in drinking water, an epidemiological investigation was conducted with 155 individuals living in a village where the arsenic concentration in the drinking water was 969 μg/L. Blood and urine samples were collected from 66 individuals including 51 cases with skin lesions and 15 controls without skin lesions. The results showed that monomethylated arsenic (MMA), the percentage of MMA (%MMA) and the ratio of MMA to iAs (MMA/iAs) were significantly increased in patients with skin lesions as compared to controls, while dimethylated arsenic (DMA), the percentage of DMA (%DMA) and the ratio of DMA to MMA (DMA/MMA) were significantly reduced. The percent DMA of individuals with the Ala/Asp genotype of glutathione S-transferase omega 1 (GSTO1) was significantly lower than those with Ala/Ala. The percent MMA of individuals with the A2B/A2B genotype of arsenic (+ 3 oxidation state) methyltransferase (AS3MT) was significantly lower than those with AB/A2B. The iAs and total arsenic (tAs) content in the urine of a Tibetan population were significantly higher than that of Han and Hui ethnicities, whereas MMA/iAs was significantly lower than that of Han and Hui ethnicities. Our results showed that when exposed to the same arsenic environment, different individuals exhibited different urinary arsenic metabolism patterns. Gender and ethnicity affect these differences and above polymorphisms may be effectors too.     

The metabolism of 7-ethoxycoumarin in primary maintenance cultures of adult rat hepatocytes

Summary 7-Ethoxycoumarin is metabolized to 7-hydroxycoumarin in short-term (1–4 days) maintenance cultures of adult rat hepatocytes. The 7-hydroxycoumarin is predominantly found as the sulphate and glucuronic acid conjugates. This pattern of metabolism is very similar to that observed with freshly-isolated rat hepatocytes and suggests that the culture system may be of value in studying the metabolism of novel chemicals designed for human therapeutic use. The specific activity of microsomal monooxygenase activity falls by 50–60% during 4 days in culture. This is not reflected by the sulphate and glucuronic acid conjugation pathways which are retained at normal levels throughout the entire 4-day culture period.     

Interspecies differences in acetaminophen sensitivity of human, rat, and mouse primary hepatocytes

Most of the experiments studying acetaminophen (APAP) induced hepatotoxicity were performed using moue as model specie, right because its high sensitivity. While the toxic responses can be called forth easily in mice, the human relevancy of these results is questionable. In this study human, rat, and mouse primary hepatocytes were treated with increasing concentrations of APAP, and cell viability was measured by MTT cytotoxicity assay. Pronounced interspecies differences were obtained in cell viability following 24 h of APAP treatment starting at 24 h after seeding (EC₅₀: 3.8 mM, 7.6 mM, and 28.2 mM, in mouse, rat, and human hepatocyte culture, respectively). The longer time of culturing highly increased the resistance of hepatocytes of all species investigated. In rat hepatocyte culture EC₅₀ values were 6.0 mM, 12.5 mM, and 18.8 mM, when starting APAP treatment after 24, 48, and 72 h of seeding. Although N-acetylbenzoquinoneimine, a minor metabolite of APAP, which is mainly formed by CYP2E1 at high APAP concentration in every species studied, is thought to initiate the toxic processes, no correlation was found between CYP2E1 activities and hepatocyte sensitivity of different species. We conclude that the toxicity induced by APAP overdose highly depends on the animal model applied.     

Interindividual variation in the capacity-limited renal glucuronidation of probenecid by humans

A dose of 1,000 mg probenecid was administered orally to 14 human volunteers in order to quantify the maximal rate of formation and excretion of probenecid acyl glucuronide in the urine. Probenecid showed dose-dependent pharmacokinetics. Plasma protein binding of probenecid was high, being somewhat higher in males (90.7±1.4%) than in females (87.9±1.4%; p=0.0019). It was shown that probenecid is metabolized by cytochrome P-450 into at least two phase I metabolites. Each of the metabolites accounted for less than 12% of the dose administered; the main metabolite probenecid acyl glucuronide, representing 42.9±13.2% of the dose, was only present in urine and not in plasma. The renal excretion rate-time profile of probenecid acyl glucuronide showed a plateau value in the presence of an acidic urine pH. This plateau value was maintained for about 10 h at the dose of 1,000 mg. The height of the plateau value depended on the individual and varied between 250 and 800g/min (15–50 mg/h). It was inferred that probenecid acyl glucuronide is formed in the kidney during blood-to-lumen passage through the tubular cells. We conclude that the plateau value in the renal excretion rate of probenecid glucuronide reflects itsV _max of formation.     

Effects of brazilin on glucose metabolism in primary cultured rat hepatocytes

In order to investigate the cellular mechanisms of hypoglycemic action of brazilin, hepatocyte monolayer culture was introduced and, glycogen synthesis rate and insulin binding were measured as parameters. Glycogen synthesis and insulin sensitivity were remarkably augmented by the treatment of brazilin. Brazilin slightly increased insulin binding. Scatchard analysis revealed that this increase in insulin binding was not due to increase in the binding capacity but in binding affinity. These results suggest that the augmentation of hepatic glycogenesis and insulin sensitivity by brazilin may play an important role in the improvement of hyperglycemia.     

Differential epigenetic effects of chlorpyrifos and arsenic in proliferating and differentiating human neural progenitor cells

Understanding the underlying temporal and mechanistic responses to neurotoxicant exposures during sensitive periods of neuronal development are critical for assessing the impact of these exposures on developmental processes. To investigate the importance of timing of neurotoxicant exposure for perturbation of epigenetic regulation, we exposed human neuronal progenitor cells (hNPCs) to chlorpyrifos (CP) and sodium arsenite (As; positive control) during proliferation and differentiation. CP or As treatment effects on hNPCs morphology, cell viability, and changes in protein expression levels of neural differentiation and cell stress markers, and histone H3 modifications were examined. Cell viability, proliferation/differentiation status, and epigenetic results suggest that hNPCs cultures respond to CP and As treatment with different degrees of sensitivity. Histone modifications, as measured by changes in histone H3 phosphorylation, acetylation and methylation, varied for each toxicant and growth condition, suggesting that differentiation status can influence the epigenetic effects of CP and As exposures.     

三氧化二砷逆转乳腺癌细胞株MCF-7/ADM多药耐药的研究

目的探讨三氧化二砷(As2O3)体外逆转人乳腺癌细胞多药耐药性的作用及机制。方法采用MTT法检测As2O3的细胞毒作用和处理前后耐药细胞对化疗药物的敏感性,用流式细胞仪检测细胞内阿霉素浓度,通过RT-RCR检测MDR1基因的表达。结果 As2O3在0.25mg/L以下剂量时对MCF-7和MCF-7/ADM耐药细胞株的抑制率均小于15%,半数抑制率(IC50)分别为1.01m/L和1.28mg/L,无细胞毒剂量0.2mg/L的As2O3能部分逆转MCF-7/ADM细胞对阿霉素的耐药性。同时无细胞毒剂量0.2mg/L的As2O3能使MCF-7/ADM细胞内阿霉素浓度明显增加,MDR1表达下降。结论 As2O3具有体外部分逆转人乳腺癌细胞多药耐药性的作用,可能与增加细胞内药物积累、下调MDR1表达有关。     

Human variation and CYP enzyme contribution in benfuracarb metabolism in human in vitro hepatic models

Human responses to the toxicological effects of chemicals are often complicated by a substantial interindividual variability in toxicokinetics, of which metabolism is often the most important factor. Therefore, we investigated human variation and the contributions of human-CYP isoforms to in vitro metabolism of benfuracarb. The primary metabolic pathways were the initial sulfur oxidation to benfuracarb-sulfoxide and the nitrogen-sulfur bond cleavage to carbofuran (activation). The K_m, V_max, and CL_int values of carbofuran production in ten individual hepatic samples varied 7.3-, 3.4-, and 5.4-fold, respectively. CYP2C9 and CYP2C19 catalyzed benfuracarb sulphur oxidation. Carbofuran formation, representing from 79% to 98% of the total metabolism, was catalyzed predominantly by CYP3A4. The calculated relative contribution of CYP3A4 to carbofuran formation was 93%, while it was 4.4% for CYP2C9. The major contribution of CYP3A4 in benfuracarb metabolism was further substantiated by showing a strong correlation with CYP3A4-selective markers midazolam-1′-hydroxylation and omeprazole-sulfoxidation (r = 0.885 and 0.772, respectively). Carbofuran formation was highly inhibited by the CYP3A inhibitor ketoconazole. Moreover, CYP3A4 marker activities were relatively inhibited by benfuracarb. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro activation of benfuracarb and that CYP3A4-catalyzed metabolism is the primary source of interindividual differences.     

三氧化二砷对三阴性乳腺癌细胞增殖及凋亡的抑制作用

目的探讨三氧化二砷对人三阴性乳腺癌细胞HCC1937的抑制作用。方法体外培养人乳腺癌HCC1937细胞,利用MTT法及倒置荧光显微镜观察三氧化二砷对人乳腺癌HCC1937细胞生长的抑制作用。结果不同浓度的三氧化二砷均可抑制人乳腺癌HCC1937细胞的增殖,促进其凋亡。三氧化二砷20μg/m1对肿瘤细胞作用72h,其生长抑制率达80.51%。结论三氧化二砷可有效抑制人乳腺癌HCC1937细胞生长,且呈时间、剂量依赖关系。     

Protein binding capacity in vitro changes metabolism of substrates and influences the predictability of metabolic pathways in vivo

Large numbers of lipophilic molecules are attached to fractions of serum protein, e.g. albumin, in vivo. Cell culture medium of most in vitro hepatocyte models for the prediction of metabolism does not contain albumin. Consequently, in vitro availability and metabolism of substrates could differ significantly from the in vivo situation. The influence of albumin on the in vitro metabolism was tested on a new lipophilic compound.

Methods: Primary human and rat hepatocytes were cultured in a collagen sandwich configuration and incubated with ¹⁴C-labeled compound X127 that is known to exhibit a high propensity to bind to plastic surfaces. Groups contained either 1% (w/v) BSA or none. Substrates as well as metabolism products were determined with radio-HPLC and radioactivity levels in the medium were recorded.

Results: Quantitative differences were seen in the distribution of the compound in BSA and non BSA containing groups, thus indicating a substantial binding of the compound to polystyrol surfaces of cell culture dishes. Metabolic radio-HPLC profiles showed different patterns after 24 h of incubation between the two species as well as between the BSA- and non-BSA groups within the species.

Conclusions: With addition of albumin the adherance of lipophilic substrates and metabolites to cell culture dish surfaces can be neutralized and in vitro systems can more closely mimic the in vivo situation.     

Study of inorganic arsenic methylation by rat liver in vitro: relevance for the interpretation of observations in man

The biotransformation of inorganic arsenic by rat liver in vitro leads to the production of a monomethylated and a dimethylated arsenic derivative, measured by flameless atomic absorption as monomethylarsonic (MMA) and dimethylarsinic (DMA) acids respectively. The methylating activity is localized in the cytosol and accepts only As³⁺ as substrate. Its optimum pH lies between 7.5 and 8.0, and reduced glutathione (10^–2M) is required for full activity. S-Adenosylmethionine is the essential methyl group donor and corrinoïd derivatives act synergistically.An excess of substrate and the addition of mercuric ions prevent the formation of the dimethylated arsenic derivative without affecting that of the monomethylated compound. This indicates that two different enzymatic activities are involved in the methylation of inorganic arsenic in mammals. Previous observations in man (Buchet et al. 1981 b, 1984) and the results of the present study suggest that DMA production results from the subsequent methylation of the MMA precursor, although the possibility that metabolites are also produced by two completely independent pathways cannot yet be conclusively rejected. The kinetics of MMA and DMA production provide an explanation for the observations that in volunteers given increasing amounts of As³⁺, the urinary excretion of DMA levels off faster than that of MMA and in patients acutely intoxicated with As³⁺, several days may elapse before DMA becomes the preponderant metabolite. The results of the present study also suggest that the reduction of MMA production associated with an increased synthesis of DMA found in patients with liver diseases given a standard dose of As³⁺ might be due to a reduction of As³⁺ uptake by the liver cells.