Selenite metabolites react in vivo with sulfobromophthalein (BSP)to form cholephiuc BSP-Se metabolites

134

Poster Session 2F. Metals

p21 expression. To extend this finding, specific alteration of gene expression in each cell cycle phase following MeHg exposure was determined using the flow-based cell sorting, RNA amplification and Reverse Northern Blotting . To identify changes in the expression of others, unidentified genes in respon se to MeHg exposure, Differential Display technique was employed. Northern Blot analysis of the clones obtained confirmed an average 2.5 induction of mitochondrial cytochrome C oxidase subunits I following MeHg exposure. To study mechanisms of cell death associated with MeHg exposure, the involvement of apoptosis in MeHg-induced cell death was evaluated. MeHg exposure resulted in an increase in both apoptotic and necrotic cell death , and the degree of apoptosis that occurred was highly dependent on MeHg exposure duration and concentration. At an equatoxic dose, the degree of apoptosis in MeHg-treated cells was approximately three times lower than cells treated with the apoptosis control agent colchicine. Together, our studies here present initial evidence regarding the mechanism of cell cycle arrest and cell death elicit by MeHg. The study may help understand the mechanism of cell loss associated with MeHg exposure during development. (Supported by EPA Grant No. R825358-0l·0, and DOE Cooperative Agreement #DE-FCOl-95EW55084)

I P2F162 I

INFLUENCE OF MINERAL DRINKING WATER ON THE CYTOTOXIC EFFECTOF RADlo-MERCURY EXPOSURE WITH LOWDOSE

S.D. lvanov - , V.Yu. Nechiporenko, A.M. Malov. Central Research Institute ofRoentgenology & Radiology, Sr. Petersburg, Russia The majority of the environmental injuring influences upon living organisms is due to the action of low-doses chemical factors . and radiation with low doses is able to modify significantly their cytotoxic effects on somatic cells. At the same time, the correction of hazard for the organism after such combined actions of toxicants is a problem. The purpo se of the present investigation is to study a recovery effect of mineral drinking water after radio-mercury exposure with low dose, which presents hazard to the health. The experiments were performed in the 3-month-old female rats receiving with drinking water Hg2+-ions in concentration I rnkg/l during I month before and I month after single gamma-irradiation with 25 cGy dose . A low mineralized Ca-,Mg-containing natural spring water was investigated as a miner water. There were studied haematological parameters, DNA content per leucocyte, and mercury concentration in blood (by nonflame atomic-absorption photometry). The introduction of mineral water resulted in increase (p < 0.05) of haemoglobin content and erythrocyte amount in blood of irradiated animals, in comparison with the corresponding control, at the early stage of postradiation restoration (7 days). The effect was constant within I month after irradiation. Monitoring of Hg-ions in blood did not reveal significant differences between any groups, apparently, as a result of low concentration of this agent in drinking watcr (at the permissible level). At the same time. the biotesting allowed to reveal a medical effect of mineral water after injuring radiation-mercury low dose exposure. In I month after irradiation an increase of lymphocyte amount was observed in blood of rats, receiving mercury, when only mineral water was introduced, but not in irradiated controls.

IP2F163I GLUTATHIONE-DEPENDENT BILIARY EXCRETION

OF SELENIUM IS ENHANCED PARADOXICALLY BY SULFOBROMOPHTHALEIN

A. Gyurasi cs .' , P. PeIjesi 2 , Z. Gregus' . I Depts. of Pharmacology; 2Medical Chemistry; Unh~ Med. School, Pees, Hungary Biotransformation of selenite involves both reactions with glutathione (GSH) and methylations. Therefore. the role of GSH,

methylation and the hepatobiliary GSH transporter was investigated in the biliary excretion of selenium in rats injected with 75Se-sodium selenite (1-10 ILmollkg, iv). Biliary output of selenium exhibited apparent capacity limitation with an approx imately 3 nmol/kg -min maximal rate and a dose-related decline in the fractional excretion. HPLC analysis indicated absence of selenite and presence of selenodiglutathione (and/or its hydrolysis products) as the predominant metabolite( s) of selenite in bile. Depletion of hepatic glutathione by D,L-buthionine-[S,R]-sulfoxirnine or diethyl maleate decreased selenium excretion into bile by 60 and 80%, respectively. In contrast, inhibitors of methylation, i.e. periodate-oxidized adenosine or ethionine, doubled the rate of biliary selenium excretion. While indocyanine green, an inhibitor of hepatobiliary GSH transport, failed to influence biliary selenium output , sulfobromophthalein (SSP), another inhibitor of this sort, dramatically enhanced it. This effect was found to be a function of the dose of both selenite and BSP. The degree of BSP-induced enhancement of the selenium excretion rate gradually increased with elevation of the selenite dose, approaching 20-fold at 10 JLmollkg selenite. In contrast, the stimulatory effect of BSP on biliary selenium output gradually lessened with elevation the BSP dose above 100 ILmollkg. In summary, this study has revealed that selenite appears in bile most likely as selenodiglutathione and that biliary selenium excretion in selenite-injected rats is dependent on the availability of hepatic GSH, counteracted by methylation, insensitive to inhibitors of the hepatobiliary GSH transporter, and enhanced paradoxically by BSP several fold . The mechanism of this unexpected effect is explored in the adjoining presentation (Gregus et all . (Supported by the Ministry of Welfare and the National Scientific Research Fund, Hungary.)

IP2F164I

SELENIUM STATUS IN THE BLOOD OF STUDENTS AND SEA FOODFOR RECOMMENDATION OF SUPPLEMENTATIONINILLENESS

A. Sukumar v", A. Muhuntha, R. Subramanian. I Tagore Ans

College, Pondicherry and JawaharlalNehru University, New Delhi. India Selenium is a toxic element in excess concentration, but also an essential trace element known for antioxidant activities . Low dietary intake is implicated in many chronic disease s. Information is, therefore, required to quantify the Se status in man and dietary sources. The samples of blood of students and edible portion of sea food were analysed fluorometrically for Se levels. The recoveries (95%-98%), precision studies (less than 6%) and analysis of Standard Reference Materials demonstrated reliability and accuracy of analytical methods . The mean Se levels (mglkg), 0.08 ± 0.03 , 0.18 ± 0.02, 0.9 ± 0.2,0.59 ± 0.08, 0.78 ± 0.1 and 0.3 ± 0.15 are observed respectively in the blood of students (n = 180), crab , cuttlc fish, shrimp, mussel and 8 species of fish (n = 10). Se levels are nol significantly different due to sex. The other factors influencing Se levels are considered. while biomonitoring of environmental status of Se is carried out. The values indicate marginal to deficient Se levels in the blood of students and dietary supply through sea food from the region of Mahe , a West Coastal Town of South India . Se supplementation may be cons idered in the treatement of various diseases .

I P2F165I SELENITE METABOLITES REACT IN VIVOWITH SULFOBROMOPHTHALEIN (BSP)TO FORM CHOLEPHIUC BSP-SeMETABOLITES

Z. Gregus' , A. Gyurasics *1, P. Perjesi 2 . JDepts. of Pharmacology; 2MedicaI Chemistry, Univ. Med. School, Pees, Hungary This work was intended to explore the mechan ism whereby BSP, an electrophilic and cholephilic organic acid. increases the biliary excretion of selenium in rats injected with 75Se-labelled sodium

PosterSession 2F. Metals

selenite (see Gyurasics et a1). In such animals, non-electrophilic congeners of BSP failed to enhance the hepatobiliary transport of selenium, suggesting that reaction of nucleophilic selenite metabolites formed in vivo with the injected BSP may be involved. Indeed, HPLC analysis of bile from rats receiving 75Se-selenite and BSP revealed two peaks (X and Y) that representedselenium-containing BSP metabolites. Pretreatment of rats with inhibitors of selenium methylation drastically diminished the size of peak X, indicating that production of metabolite X is dependent on formation of methylated selenium metabolites. A compound chromatographically indistinguishable from that in peak X was formed in vitro duringincubation of BSP with methylselenol, suggesting that biliary metabolite X is identical to the reaction product of BSP and selenite-derived methylselenol. Incubation of BSP with selenite in the presence of thiols, e.g., glutathione, (which can convert selenite into hydrogen selenide) resulted in product(s) chromatographically similar to the biliary selenium-containing BSP metabolite(s) of peak Y, irrespective of the nature of thiol used. Thus, biliary metabolite(s) Y may be reaction products of BSP and hydrogen selenide. Finally, BSP significantly diminished exhalation of dimethyl selenide in selenite-injected rats, purportedly because it reacted with precursors of dimethyl selenide, thatincludehydrogenselenideand methylselenol. In summary, BSP increases the biliary excretion of selenium in rats receiving selenite because it forms selenium-containing BSP metabolites that are readily transported into bile. In vivo reaction of nucleophilic selenite metabolites with electrophilic compounds may influence the fate of selenium and may contribute to the anticarcinogenic effect of this metalloid.

IP2F166I

ARSENIC(AS3+) INCREASESCELLULAR HOMOCYSTEINE (Hey) IN COBALMIN(B12) DEFICIENT HUMAN FIBROBLASTS:A MECHANISM FOR NUTRIENTfTOXICANT INTERACTION

C.R. Angle *, S.A. Swanson. University ofNebraskaMedical Center, Omaha,NE, USA AsJ+ enhances the increased proliferation of human vascularsmooth muscle cells (HVASMC) and human fibroblasts induced by Hey in B12 depleted culture of quiescent cells. To test the hypothesis that 812 deficieney and As3+ each increased intracellular Hey, the Hcy contentof humanfibroblasts wasassayedby the fluorescent detection of free and protein bound Hcy (Araki A & Sako Y, J Chromatog 1987;422:43-52). In B12 replete media (MEM plus 0.1% fetal calf serum) with 0.4-0.8 roM Hey,the protein boundHey was decreased by As3+. Glutathione was also decreased. The dose responseto As 3+ was suggestive of the essentiality of trace concentrations As3+ for the methylation of S-adenosyl Hcy to S-adenosyl methionine. In B12 deficient media, As3+ evoked a significant increase of intracellular Hcy consistent with the increased cellular proliferation found in response to Hcy plus As3+. Glutathione was increased suggesting that glutathione utilization by As3+ may be impaired by 812 deficiency. The cell culture responses indicate a mechanism for an apparent nutrient toxicant interaction. The methylation demands of excessive AsJ + may be synergistic with the nutritional and genetic causes of hyperhomocysteinema resulting in the severevascularand dermal proliferation typical of malnourished populations in areas of epidemic arsenicism.

IP2F167 I DETERMINATION OF HARDEROPORPHYRIN A BIOMARKERFOR ARSENICEXPOSURE

1.C.Ng *, L. Qi, B. Chiswell, M.R Moore. NationalResearch Centrefor Environmental Toxicology, The University ofQueensland, Brisbane, Australia An improved HPLC method has been established for the measure-

135

ment of harderoporphyrin (HP) in the harderian glandof rodents. HP degrades to protoporphyrin IX (PP) in vitro at room temperature. It is essential to carry out the analytical procedures in cold temperature. Significantly increasedconcentrations of HP werefoundin harderian glands of rats. Groups of femaleWistarrats were dosed by oral gavage witha solutionof sodiumarseniteat 0, 0.5 or 5.0 mg Aslkgbody weight, or a slurry of arsenic-contaminated soil at equivalent dose rates. The animals were sacrificed 96 hours after dosing, harderian glands removed and stored at -80"C until analysis. The ratio of HPIPPincreased from 1.04 ± 0.07 (n = 8) for the controlrats to 1.33 ± 0.10 (5) and 1.48 ± 0.09 (5) for rats given 0.5 mg Aslkg and 5.0 mg Aslkg body weight respectively. Significant increases were also observedin some of the soil dosed groups. Our results suggest that the alteration of porphyrins profile in the harderian glands of rodents is highly sensitive to As exposure and can be used as early bioindicator for arsenic exposure in rats and in health risk assessment for other sentinel species at arsenic contaminated sites.

IP2F168I

BIOAVAILABILITY OF ARSENIC IN CONTAMINATED MEDIA

S.W. Casteel *' , L.D. Brown", RP. Cowart", t .w Pace' , E. Hoffman'", G.M. Henningserr'. C.P.Weis2 . J Universityof Missouri, Columbia, MO; lU.S. EPARegion VIII, Denver, CO, USA Assessment of human health risks resulting from oral exposure to arsenic requires knowledge of its enteric absorption fraction. Reliable site-specific data on arsenic bioavailability from environmental mediadecreases the uncertainty in human health risk estimates. Immatureswine were usedas a plausiblemodelfor children. Groupsof 4-to-5 pigs wereorallydosedfor 15days witheitherarsenic-containing media or sodium arsenate(NaAs) given orally or intravenously. The amount of absorbed arsenic was assessed by measuring the amount excreted in urine. Urinary arsenic excretion was a linear function of dose and was independent of time after day 5. Therefore, the urinary excretion fraction (UEF) was estimated by the slope of the best-fit regression line through the dose-response data after 5 days of dosing. Estimates of relative bioavailability (RBA(x) = UEF (x)IUEF (NaAs, oral) of arsenic in different media ranged from 1% to 70%. Based on the ratio of UEF of NaAs given orally to that for NaAs IV, oral NaAs given to semi-fasted pigs is about 40% absorbed. RBA resultsfor differenttest media supportthe view that arsenic in mine waste and soil is not as well absorbed as soluble arsenic(NaAs), thereforeuse of defaulttoxicityfactors for assessing humanhealthrisk will lead to an overestimate of hazard. (Supported by U.S. EPA)

IP2F169 I

IS THE USE OF 24 HOUR URINESAMPLES AND CREATININE ADJUSTMENTREQUIRED FOR ANALYSISOF INORGANIC ARSENIC IN URINEIN POPULATION STUDIES?

M.R Sim *. A.L. Hinwood, DJ. Jolley, E.B. Bastone, U. McNeil. DepartmentofEpidemiology and Preventive MedicineMonash University, Australia Background and Objectives: 24 hr urine samples are used to measure short term absorption of inorganic arsenic as they are thoughtto provide a more reliable result than spot samples. However, compliance with 24 hr urine samples can be low in population-based studies. This study aimed to determine the relationship between24 hr urine and spot samples and also whethercreatinineadjustment is important for measurement of urinary arsenicconcentrations. Materials and Methods: Urine samples were collected from 206 peoplelivingin areaswithhighenvironmental arsenicconcentrations and a comparison area with low arsenic concentrations. Each subject