Concentration- and time-dependent genomic changes in the mouse urinary bladder following exposure to arsenate in drinking water for up to 12 weeks.

Inorganic arsenic (As(i)) is a known human bladder carcinogen. The objective of this study was to examine the concentration dependence of the genomic response to As(i) in the urinary bladders of mice. C57BL/6J mice were exposed for 1 or 12 weeks to arsenate in drinking water at concentrations of 0.5, 2, 10, and 50 mg As/l. Urinary bladders were analyzed using gene expression microarrays. A consistent reversal was observed in the direction of gene expression change: from predominantly decreased expression at 1 week to predominantly increased expression at 12 weeks. These results are consistent with evidence from in vitro studies of an acute adaptive response that is suppressed on longer exposure due to downregulation of Fos. Pathways with the highest enrichment in gene expression changes were associated with epithelial-to-mesenchymal transition, inflammation, and proliferation. Benchmark dose (BMD) analysis determined that the lowest median BMD values for pathways were above 5 mg As/l, despite the fact that pathway enrichment was observed at the 0.5 mg As/l exposure concentration. This disparity may result from the nonmonotonic nature of the concentration-responses for the expression changes of a number of genes, as evidenced by the much fewer gene expression changes at 2 mg As/l compared with lower or higher concentrations. Pathway categories with concentration-related gene expression changes included cellular morphogenesis, inflammation, apoptosis/survival, cell cycle control, and DNA damage response. The results of this study provide evidence of a concentration-dependent transition in the mode of action for the subchronic effects of As(i) in mouse bladder cells in the vicinity of 2 mg As(i)/l.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water.

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder >>> skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.

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.

In vitro dermal absorption of flame retardant chemicals.

Flame retardant chemicals may be used in furniture fabric in the future to reduce the flammability of the fabric. As a part of the process to evaluate the potential for exposure to these chemicals, this study examined the in vitro dermal absorption of two flame retardant chemicals. The chemicals were [14C]decabromodiphenyl oxide (DBDPO) and [14C]tris-(1,3-dichloro-2-propyl)phosphate (TDCP). Skin from the adult hairless female mouse (SKH1) was removed and mounted in flow-through diffusion cells. The chemicals, at three dose levels (DBDPO: 6, 30 and 60 nmol; TDCP: 20, 100 and 200 pmol), were applied in a volatile vehicle (tetrahydrofuran for DBDPO; acetone for TDCP) to the skin. Fractions of receptor fluid, pumped below the skin, were collected over a 24-h period. The skin was washed with solvent (tetrahydrofuran for DBDPO; ethanol for TDCP) to remove unabsorbed chemical 24 h after application. The receptor fluid, skin wash and skin were analyzed for chemical-derived radioactivity. The skin from the high-dose group of both chemicals, and the receptor fluid from TDCP high-dose samples, were analyzed for parent compound and metabolites by HPLC. The 24-h cumulative percent of the dose of DBDPO in the receptor fluid was very low (0.07-0.34%). The applied dose of DBDPO detected in the skin ranged from 2 to 20%. The lowest dose of DBDPO had the highest percentage of the dose (20%) in the skin. The major portion of the applied dose was removed by washing the skin 24 h after application of DBDPO, and ranged from 77 to 92%. HPLC analysis of homogenate extract prepared from the high-dose of DBDPO-treated skin showed the presence of DBDPO and a minor unknown peak. TDCP was readily detected in the receptor fluid; 39-57% of the applied dose of TDCP was in the receptor fluid by 24 h. The solvent wash removed 11-25% of the dose from the skin and 28-35% remained in it. HPLC analysis of the skin homogenate extract and receptor fluid extract from the TDCP high-dose treated samples showed the presence of parent compound and a minor unknown peak. TDCP more readily penetrated hairless mouse skin and diffused into the receptor fluid than DBDPO. TDCP has a lower molecular weight and log octanol:water partition coefficient than DBDPO. The differences in the physico-chemical properties of these two chemicals most likely explains their dissimilar absorption through hairless mouse skin.

A concise review of the toxicity and carcinogenicity of dimethylarsinic acid.

Dimethylarsinic acid (DMA) has been used as a herbicide (cacodylic acid) and is the major metabolite formed after exposure to tri- (arsenite) or pentavalent (arsenate) inorganic arsenic (iAs) via ingestion or inhalation in both humans and rodents. Once viewed simply as a detoxification product of iAs, evidence has accumulated in recent years indicating that DMA itself has unique toxic properties. DMA induces an organ-specific lesion--single strand breaks in DNA--in the lungs of both mice and rats and in human lung cells in vitro. Mechanistic studies have suggested that this damage is due mainly to the peroxyl radical of DMA and production of active oxygen species by pulmonary tissues. Multi-organ initiation-promotion studies have demonstrated that DMA acts as a promotor of urinary bladder, kidney, liver and thyroid gland cancers in rats and as a promotor of lung tumors in mice. Lifetime exposure to DMA in diet or drinking water also causes a dose-dependent increase in urinary bladder tumors in rats, indicating that DMA is a complete carcinogen. These data collectively suggest that DMA plays a role in the carcinogenesis of inorganic arsenic.

Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration.

Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3-4/time point) were administered 1.11 or 111 mg/kg of DMA (1 microCi of [14C] or unlabeled) intravenously and killed serially (5-480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration-time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1-20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t1/2beta and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ.

Strain-dependent disposition of inorganic arsenic in the mouse.

Recent studies have suggested that polymorphisms in the methylation of inorganic arsenic (iAs) exist in animals and humans. Methylation of iAs is an important step in the elimination of arsenic. The objective of this study was to examine whether there are differences in iAs disposition, and hence methylation, between three strains of mice. Ninety-day-old female mice (strains: C3H/HeNCrlBR, C57BL/6NCrlBR, and B6C3F1/CrlBR) were administered [73As]arsenate or [73As]arsenite orally at dose levels of 0.5 or 5.0 mg As/kg. Another group of mice were administered [73As]arsenate (5.0 mg As/kg) intraperitoneally (i.p.). Disposition of [73As] was assessed by whole-body counting, and analysis of urine, feces and tissues for radioactivity. Urine was analyzed by chromatography for arsenic metabolites. Several strain- and dose-related effects in the disposition of [73As] were observed with both arsenicals. After oral administration, the clearance of [73As]arsenate, measured by whole-body counting, was dependent on the strain. However, because there was no strain dependence on clearance of [73As]arsenate administered i.p., the effect after oral administration may be due to a difference in absorption of arsenate between the strains. With increased oral dose of arsenate and arsenite, the clearance of [73As] was slower and there was higher tissue retention of [73As]. The percentage of metabolites excreted in urine also was affected by the administered dose. With increased dose, the percentage of arsenite and monomethylarsonic acid were significantly increased, and dimethylarsinic acid decreased. However, our results suggest there is no overall difference between these strains of mice with respect to disposition of iAs. A better understanding of the role of phenotype in the disposition and toxicity of iAs would reduce the uncertainty in arsenic risk assessment.

Dose-dependent effects on the disposition of monomethylarsonic acid and dimethylarsinic acid in the mouse after intravenous administration.

The organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are the primary metabolites of inorganic arsenic, a known human carcinogen. The objective of this study was to examine if dose would affect the excretion and terminal tissue disposition of MMA and DMA in the mouse. 14C-MMA (4.84 and 484 mumol/kg) and -DMA (8.04 and 804 mumol/kg) were administered to female mice via the tail vein. The mice were placed in metabolism cages for collection of urine (1, 2, 4, 8, 12, and 24 h) and feces (24 h). The animals were then sacrificed at 24 h and tissues were removed and analyzed for radioactivity. The urine was also analyzed for parent compound and metabolites. Urinary excretion of MMA- and DMA-derived radioactivity predominated over fecal excretion. Dose did not affect the overall urinary excretion of both compounds. However, fecal excretion was significantly lower in the low-dose MMA-treated animals as opposed to in the high-dose group, whereas in the high-dose DMA-treated group excretion was lower than in the low-dose DMA group. The retention of radioactivity was low (< 2% of dose) and the distribution pattern similar for both compounds, with carcass > liver > kidney > lung. The concentration of radioactivity (% dose/g tissue) was greater in kidney than in liver, lung, and blood for both compounds. The distribution and concentration of MMA-derived radioactivity was significantly greater in the liver and lung of the high-dose group. The MMA-treated animals excreted predominantly MMA in urine and lower amounts of DMA (< 10% of the dose). The percentage excreted as DMA was significantly higher in the low-dose MMA group. In the urine of DMA-treated animals, an unstable metabolite and the parent compound were detected. Overall, it appears the dose of organic arsenical administered has a minimal effect on its excretion and terminal tissue disposition in the mouse. The rapid elimination and low retention of MMA and DMA explain in part their low acute toxicity.

In vivo disposition of p-substituted phenols in the young rat after intraperitoneal and dermal administration.

The objective of this study was to examine the 120-hr disposition of phenol and four p-substituted congeners after ip and dermal administration in the 29-day-old female rat. The dermal absorption was very high (66-80% of the dose) for phenol, cyanophenol, heptyloxyphenol and nitrophenol, but minimal for hydroxybenzoic acid (2%). The major portion of the dose for all of the phenols not absorbed dermally in 24 hr was washed from the skin. Only minor amounts (1-2%) were detected in the treated skin at 120 hr. Urinary excretion was the predominant means of elimination for these phenols and occurred primarily within 24 hr after dermal and ip administration. However, the excretion of heptytoxyphenol after administration by both routes differed from that of the other compounds, with more of it detected in the faeces. The profile of metabolites in urine (collected at 12-24 hr) from the animals dermally treated with phenol, cyanophenol, heptyloxyphenol and nitrophenol showed only peaks that eluted earlier than the parent compound, which suggests that conjugates or more polar metabolites were formed and excreted. The difference in dermal absorption between hydroxybenzoic acid and the other phenols may be due to potential ionization of the p-substituted carboxylic acid group of hydroxybenzoic acid. This suggests that, at least for the phenols examined in this study, physicochemical characteristics other than just lipophilicity can affect in vivo dermal absorption.

Influence of dietary selenium on the disposition of arsenate in the female B6C3F1 mouse.

Interactions between arsenic (As) and selenium (Se) at the metabolic level are multifaceted and complex. These interactions are of practical significance because populations in various parts of the world are simultaneously exposed to inorganic As in drinking water and Se mainly in the diet at varying levels. The primary goal of this study was to investigate whether differing dietary Se status would alter the profile of urinary metabolites or their time course for elimination after exposure to arsenate [As(V)]. Weanling female B6C3F1 mice were maintained for 28 d on either a control diet of powdered rodent meal sufficient in Se (A, 0.2 ppm) or Torula yeast-based (TYB) diets deficient (B, 0.02 ppm Se), sufficient (C, 0.2 ppm Se), or excessive (D, 2.0 ppm Se) in Se; mice then received by oral gavage 5 mg (As)/kg as sodium [73As] arsenate. The time course for elimination of total arsenic and metabolites in urine was measured over a 48-h period, and total arsenic was determined in feces and tissues at 48 h. Mice on the Se excess diet excreted a significantly higher percentage of urinary As as inorganic As, with a significantly decreased ratio of organic to inorganic As compared to Se-sufficient mice, suggesting that As methylation was decreased. Mice on the Se-deficient diet appeared to eliminate As(V), arsenite, and dimethylarsinic acid (DMA) in urine more slowly than Se-sufficient mice; however, further studies are required to confirm this finding. Mice on the Se-sufficient meal diet (A) excreted significantly less (by percent) arsenate-derived radioactivity in urine and more in feces compared to mice on the Se-sufficient TYB diet (C), with total elimination being similar for both groups. This indicates that mice on the meal diet absorbed significantly less As(V) than mice on the TYB diet, and this may be due to more fiber or "bulk" in the meal diet. This finding emphasizes the importance of considering dietary composition when interpreting and comparing As disposition studies. Overall this study provides suggestive evidence that dietary Se status alters As metabolism and disposition. This indicates that dietary Se status may be an issue that should be considered in the design and interpretation of epidemiologic studies.

Assessment of light scatter by nucleoids as a rapid predictive assay of radiosensitivity.

A study has been made of the practicality of using the assay of light scatter by nucleoids as a rapid predictive test of cellular radiosensitivity. With this technique the effect of irradiation on DNA organization is measured using flow cytometry after staining irradiated nucleoids with a high concentration of ethidium bromide. Damaged nucleoids fail to respond to the ethidium bromide-induced contraction and scatter more forward-angle light than less damaged nucleoids. Seventeen different cell lines were assessed using a single lysis condition and radiation dose. Significant differences in the levels of radiation-induced forward-angle light scatter by nucleoids were seen between CHO cells and cells of two radiosensitive mutant cell lines (xrs-6, EM9), and between cells of two ovarian carcinoma lines that showed marked differences in radiosensitivity measured using a clonogenic assay. However, other cell lines which differed in clonogenic radiosensitivity showed similar forward-angle light scatter by nucleoids. When all 17 cell lines were included in the analysis, there was no correlation between measurements of radiosensitivity by assays of clonogenicity and light scatter by nucleoids. In addition, although intraexperimental variation was small, the level of interexperimental variability was only slightly smaller (coefficient of variation of 13%) than the degree of heterogeneity observed between the different cell lines (coefficient of variation of 16%). These findings support the notion for a role of nuclear structure as a determinant of intrinsic radiosensitivity for some cell lines but suggest that for others there must be additional, more dominant factors.

Subchronic dispositional and toxicological effects of arsenate administered in drinking water to mice.

Exposure to the drinking water contaminant arsenate is a daily occurrence and there are concerns that this exposure may lead to cancer. Although the acute dispositional effects of arsenate have been studied in detail, there is minimal information on the disposition and toxicological effects of it after continuous exposure. The objective of this study was to examine in mice the effect of a 4-wk treatment with arsenate administered in drinking water. Female B6C3F1 mice (3/cage) were housed in metabolism cages and given water and food ad libitum. Two groups (A, B) of mice were treated (4 cages/treatment/group) with distilled water (control, C) or water containing 0.025 mg/L (L) or 2.5 mg/L (H) arsenate. Group A was sacrificed on d 28 and plasma and urine samples were taken for determination of clinical chemistry parameters. Liver and kidney tissue samples were taken for histopathological analysis. The reduced nonprotein sulfhydryl (NPSH) content in several tissues was determined. Group B was gavaged with [73As]arsenate on d 28 and continued the arsenate drinking water exposure for 48 h. Excreta and tissues were collected and analyzed for 73As. Urine was further analyzed for arsenate and its metabolites. There were no effects on the mean daily amount of water and food consumed, whereas the mean daily urine volume excreted was significantly elevated by 10% in the H-treated animals compared to C and L. A dose-related hepatic vacuolar degeneration in the liver was observed, but no histological changes were evident in the kidney. Only clinical chemistry parameters in plasma were altered by the arsenate treatment. Glucose was significantly lower at the H dose compared to C and L, triglycerides were significantly greater in C than L and H, and creatinine was significantly greater in H than C. Hepatic NPSH content in the H animals was significantly lower than C and L animals, whereas no effects in lung and kidney were detected. The weights of liver, lung, and kidney, as well as their tissue/body weight ratios, were significantly decreased in the H animals. 73As was primarily eliminated in urine, and its elimination was not affected by dose. No effects on the 48-h 73As cumulative excretion (urine+fecal) were detected. The 73As distribution was low in amount and widely dispersed throughout the animal (< 3% of the 73As dose). The kidney had the highest 73As concentration of the tissues (0.01% 73As dose/g tissue). Dimethylarsinic acid was the major metabolite detected in urine, with lower amounts of arsenate arsenite, and monomethylarsonate. There were no differences between the treatment groups in the amount of urinary metabolites after a single dose of [73As]arsenate. Several toxicological effects were observed in animals administered arsenate in drinking water, but no changes in the disposition of this arsenical were detected at the doses used in this study.

Liberation and analysis of protein-bound arsenicals.

Protein-bound arsenicals were liberated from binding sites on liver cytosolic proteins by exposure to 0.1 M CuCl at pH 1. This method released greater than 90% of the arsenicals associated with biological matrices. Ultrafiltrates of CuCl-treated cytosols were subjected to thin-layer chromatography to speciate and quantify inorganic and methylated arsenicals. For rat liver cytosol in an in vitro methylation assay and for liver and kidney cytosols from arsenite-treated mice, most inorganic arsenic was protein bound. Appreciable fractions of the organoarsenical metabolites present in these cytosols were also protein bound. Therefore, CuCl treatment of cytosols releases protein-bound arsenicals, permitting more accurate estimates of the pattern and extent of arsenic methylation in vitro and in vivo.

Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy.

FDCP-Mix, a pluripotent murine hemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis by either drugs or withdrawal of growth factor (interleukin-3) was studied after treatment with the topoisomerase II inhibitor etoposide (0.5-4 microM). An increase in autolytic activity was the major early morphological change within the cytoplasm, with mitochondria as the main target for autolytic digestion. Despite this macroautophagy, thin sections showed a high number of mitochondria, suggesting mitochondrial proliferation as a result of drug treatment. This observation of an increase in the number of mitochondria was confirmed by flow cytometric studies of mitochondrial overall mass. Multiparameter flow cytometry of cells double stained with propidium iodide and nonyl-acridine orange gave an accurate assay for mitochondrial mass in relation to cell cycle stages. The increase in mitochondrial mass was found in all cell cycle stages. The results suggest a drug-induced proliferation of mitochondria separate from the processes involved in the doubling of mitochondrial mass during the cell cycle and a decline of mitochondria in the later stages of apoptosis.

Disposition of phenol in rat after oral, dermal, intravenous, and intratracheal administration.

The absorption and elimination of [14C]-phenol (63.5 nmol) after oral, dermal, intratracheal, or intravenous administration in rat was rapid and extensive. Urinary elimination of radioactivity predominated, with a range of 75-95% of the dose detected in urine by 72 h post-exposure. Washing the dermal site 72 h post-exposure removed 14% of the dose. Two per cent of the dose was detected in the skin. The urinary metabolites at 4 and 8 h after administration by the four routes included phenyl sulphate and lower amounts of phenyl glucuronide. Phenol was poorly retained in the body after administration by the four routes. Phenol remaining in the body was widely distributed, with accumulation primarily in the liver, lung, and kidney.

In vitro percutaneous absorption of dimethylarsinic acid in mice.

The objective of this study was to investigate the in vitro dermal absorption of [14C]dimethylarsinic acid. This organic arsenical is used as a herbicide and is a product of the mammalian metabolism of inorganic arsenic. Discs of preclipped dorsal skin were cut from adult female B6C3F1 mice and mounted in flow-through diffusion cells. HEPES-buffered Hanks balanced salt solution was used as receptor fluid. Doses of dimethylarsinic acid included 10, 100, and 500 micrograms and were applied onto the skin (0.64 cm2). Experiments (24 h) were conducted using solid compound and aqueous solution (20, 100, and 250 microliters) and soil (23 mg/cm2) as vehicles. The epidermal surface was washed at 24 h to remove compound that did not penetrate. The wash contained the greatest percentage of the dose in all experiments. Absorption of the compound into the skin and receptor fluid was observed and ranged from < 1 to 40% of the dose in experiments with the three exposure scenarios. The rank order of the various exposure conditions of dimethylarsinic acid absorption (10 micrograms) into the skin and receptor fluid was 20 microliters water > 100 microliters water > solid > 250 microliters water > soil. No dose or pH effects on absorption of dimethylarsinic acid was observed. There was also no pH effect on the partitioning of dimethylarsinic acid between 1-octanol and buffer. Short-term (1 h) exposure of dimethylarsinic acid in water followed by wash of the skin resulted in < 1% of the dose being absorbed. Thus, vehicles and duration of exposure have important roles on the in vitro dermal absorption of dimethylarsinic acid in mouse skin.

Identification of methylated metabolites of inorganic arsenic by thin-layer chromatography.

TLC on cellulose plates was used to identify methylated products of inorganic arsenic metabolism (monomethylarsonate and dimethylarsinate) in biological samples. Two solvent systems were tested: methanol-ammonium hydroxide (8:2) and isopropanol-acetic acid-water (10:1:2.5). The latter solvent system produced the most satisfactory separation of radiolabelled methylated arsenic compounds in aqueous solution, in rat liver cytosol incubated with carrier-free or 1 microM [73As]arsenite and in urine of mice given carrier-free [73As]arsenate or 5 mg of [73As]arsenate/kg per os. Oxidation of samples by hydrogen peroxide improved the separation and quantitation of monomethylarsonate in both biological matrices.

In vitro percutaneous absorption of monosodium methanearsonate and disodium methanearsonate in female B6C3F1 mice.

Percutaneous absorption of monosodium [14C]methanearsonate (MSMA) and disodium [14C]methanearsonate (DSMA) was investigated in female B6C3F1 mice from a variety of exposure vehicles, including aqueous solution, solid compound, and soil. These chemicals are the sodium salts of methanearsonic acid, an in vivo metabolite of inorganic arsenic compounds, and are present in water and soil. Permeation experiments were carried out in vitro for 24 h using previously clipped dorsal skin (area = 0.64 cm2) in flow-through cells with HEPES-buffered Hanks balanced salt solution as receptor fluid. Applied doses of 10 (15.6), 100 (156), and 500 (781) micrograms (micrograms/cm2) were studied in selected vehicles, and dermal absorption was quantitated by determining the radioactivity in the receptor fluid and skin following a skin surface wash to remove unpenetrated compound. Both MSMA and DSMA exhibited similar dermal absorption from different vehicles, and the rank order was aqueous solution > solid compound > soil. The degree of ionization of the compounds did not appear to affect their skin absorption, as both monobasic and dibasic forms penetrated mouse skin to the same extent from aqueous vehicles. An alteration in the aqueous donor volume (20, 100, and 250 microliters) did not significantly change the total absorption of the chemicals; however, larger volumes significantly prolonged the time to reach maximal permeation rates. The major portion of the absorbed dose (53% or higher) remained in the skin for both chemicals. A constant fraction of the applied dose (12.4%) was absorbed from aqueous vehicles over the entire dosage range. Absorption of the chemicals was very low (< 0.5% of the dose) from soil. Even short-term (1 h) dermal exposure to an aqueous solution containing MSMA resulted in the penetration (0.66% of the dose) of this chemical. Thus, exposure vehicles have an important role in the in vitro dermal absorption of MSMA and DSMA in mouse skin, with aqueous solutions providing the greatest absorption.

Dose-dependent disposition of sodium arsenate in mice following acute oral exposure.

The effect of dose on arsenate disposition was studied in adult female B6C3F1 mice, dosed po with 0.5 to 5000 micrograms/kg [73As]-arsenate in water. Urine was collected at 1, 2, 4, 8, 12, 24, and 48 hr and feces at 24 and 48 hr postexposure. The mice were euthanized at 48 hr and tissues were removed. Recovery of arsenate-derived radioactivity ranged from 83 to 89%; 66-79% of the dose was excreted in urine, 10-18% in feces, and < 1% remained in the tissues. Although dose had no effect on the 48-hr excretion of radioactivity, the level of radioactivity in several tissues increased significantly with dose. The urine was analyzed for arsenic metabolites by using ion chromatography to analyze for arsenate, methylarsonic acid (MMA), and dimethylarsinic acid (DMA); ion-pairing high-performance liquid chromatography was used for arsenite analysis. Arsenate elimination ranged from 3 to 15%. DMA was the predominant metabolite excreted (51-64% of dose), but no effect of dose on its elimination was detected. As the dose of arsenate increased, the amount of MMA excreted (0.1-1.0% of dose) significantly increased. At 5000 micrograms/kg arsenate, a significant increase in arsenite excretion was observed. At doses of arsenate < or = 500 micrograms/kg, peak elimination of DMA occurred within 4 hr postexposure. At the 5000 micrograms/kg dose, DMA peak elimination shifted to 8 hr and a lower amount was excreted. In addition, at the 5000 micrograms/kg dose, there was an increase of arsenate and arsenite in the 1- and 2-hr urines. These results suggest that an acute dose of arsenate can affect the metabolism of arsenicals. High doses lead to the accumulation of intermediates that are more reactive than DMA, and this response may lead to increased toxicity.

Effect of age on the in vitro percutaneous absorption of phenols in mice.

The effect of age on the in vitro dermal absorption of acetamidophenol, phenol, cyanophenol and heptyloxyphenol was examined. Skin from pre-clipped male C57BL/6N mice of ages 3, 15 and 27 months was mounted in flow-through diffusion cells. [(14)C]Phenol and its analogues (4 mug/cm(2)) were applied to the skin (0.32 cm(2)) in ethanol (5 mul) and absorption was measured under occluded conditions for 72 hr. Significant age effects on the disposition of phenol and heptyloxyphenol were observed in the penetration of compound into the receptor fluid as well as that retained in the skin. The receptor fluid content of these two compounds was significantly greater in 27-month-old mice compared with the younger animals. In addition, penetration of phenol in 15-month-old mice was significantly greater than in 3-month-old mice. However, the differences in phenol penetration were small (< 5%). The maximal flux of cyanophenol and phenol penetration were affected by age. The maximal flux for cyanophenol was significantly lower in 15-month-old mice than in 3- and 27-month-old animals. The maximal flux for phenol was significantly greater in 27-month-old mice compared with the younger animals. The overall effect of age on the in vitro dermal absorption of phenols in C57BL/6N mouse skin appears to be compound dependent.