Z. Gregus

Disposition of metals in rats: a comparative study of fecal, urinary, and biliary excretion and tissue distribution of eighteen metals.

Fecal (0-4 days), urinary (0-4 days), and biliary (0-2 hr) excretion and tissue distribution of 18 metals were examined in rats after iv administration. Total (fecal + urinary) excretion was relatively rapid (over 50% of dose in 4 days) for cobalt, silver, and manganese; was between 50 and 20% for copper, thallium, bismuth, lead, cesium, gold, zinc, mercury, selenium, and chromium; and was below 20% for arsenic, cadmium, iron methyl mercury, and tin. Feces was the predominant route of excretion for silver, manganese, copper, thallium, lead, zinc, cadmium, iron, and methyl mercury whereas urine was the predominant route of excretion for cobalt, cesium, gold, selenium, and chromium; while both excretion routes were equally important for bismuth, mercury, arsenic, and tin. Biliary excretion seems to be an important determinant for the fecal excretion of silver, arsenic, manganese, copper, selenium, cadmium, lead, bismuth, cobalt, and methyl mercury. Between 45 (silver) and 0.8% (methyl mercury) of the dosages administered of these metals was excreted into bile in 2 hr, and they exhibited high bile/plasma concentration ratios. The biliary excretion of copper, selenium, lead, and chromium did not increase proportionally with dosage, suggesting that the hepatobiliary transport of these metals is saturable. The fraction of dosage excreted into bile was independent of the dosage for silver, arsenic, manganese, bismuth, methyl mercury, mercury, gold, cesium, thallium, and tin, but markedly increased with increase in dosage of cadmium, cobalt, zinc, and iron. The latter phenomenon is probably due to saturation of hepatic (cadmium, zinc) or extrahepatic (iron) metal-binding sites. Comparison of biliary and fecal excretion rates indicates that arsenic and selenium undergo intestinal reabsorption, whereas thallium and zinc enter the feces also by non-biliary routes. Most of the metals reached the highest concentration in liver and kidney. However, there was no direct relationship between the distribution of metals to these excretory organs and their primary route of excretion.

Hepatic phase I and phase II biotransformations in quail and trout: Comparison to other species commonly used in toxicity testing☆

The ability of quail and trout to perform a number of representative phase I and phase II biotransformations was examined. To facilitate interspecies comparisons, metabolism of the same substrates was examined simultaneously under uniform conditions for rat, mouse, rabbit, guinea pig, cat, and dog. Both nonmammalian species can metabolize four representative substrates of phase I mixed-function oxidases and one substrate of epoxide hydrolase, though activity tended to be lower than that of the mammals. Important differences in the conjugative pathways were also noted. Among these differences were the quails relative deficiency in glutathione conjugation and the trouts low ability to conjugate sulfate compounds. Trout liver UDP-glucuronosyltransferase activity was remarkably high toward testosterone and bilirubin, while quail liver formed glucuronides of naphthol, p-nitrophenol, and digitoxigenin-monodigitoxoside. Also noteworthy was the high N-acetyltransferase activity of both quail and trout toward isoniazid, beta-naphthylamine, and 2-aminofluorene. Differences in substrate specificity for a given enzymatic pathway may be an indication that multiple forms of drug metabolizing systems also occur in these nonmammalian species. Observation of several hundred- or even thousand-fold differences between species in their enzyme activities for certain substrates under uniform conditions re-emphasizes the need for caution in extrapolation of xenobiotic metabolism from one species to another.

Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases☆

Abstract Differential induction with phenobarbital (PB) and 3-methylcholanthrene (3-MC) suggests at least two functionally distinct UDP glucuronosyltransferases (UDP-GT) which have different acceptor selectivities. One form is induced by 3-MC and preferentially conjugates group 1 acceptors, such as p -nitrophenol and 1-naphthol. Another UDP-GT is induced by PB and glucuronidates group 2 aglycones, morphine and chloramphenicol. To further study this functional heterogeneity, male Sprague-Dawley rats were pretreated with the following microsomal enzyme inducers: 7,8-benzoflavone (BF); benzo( a )pyrene (BP); 3-MC; 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD); butylated hydroxyanisole (BHA); isosafrole; PB; pregnenolone-16α-carbonitrile (PCN); trans -stilbene oxide (TSO). The effect of induction on UDP-GT activity was determined with nine acceptors. Conjugation of group 1 aglycones, naphthol and p -nitrophenol, was increased by 3-MC (185 and 80%, respectively) whereas PB was ineffective. Conjugation of group 2 acceptors, morphine and chloramphenicol, was stimulated by PB (120 and 250%, respectively) while 3-MC had little effect. BP and TCDD enhanced glucuronidation of group 1 aglycones. ISF and TSO induced conjugation of both acceptor groups but were more effective for group 2. BF and BHA had negligible effects on UDP-GT activity. Since glucuronidation of valproic acid was increased only by PB and TSO treatment, this aglycone is probably a group 2 acceptor. Conjugation of digitoxigenin-monodigitoxoside (DIG) was stimulated by PB (200%) and PCN (1200%). PCN did not induce glucuronidation of group 1 acceptors but did have a slight effect on group 2 aglycones (130 and 40% for chloramphenicol and morphine, respectively). The 12-fold increase in DIG conjugation by PCN pretreated rats suggests that PCN may induce another group (form) of UDP-GT which preferentially glucuronidates DIG. Differential induction of UDP-GT activities within each group of acceptors indicates possible additional heterogeneity of the transferase.

Effect of microsomal enzyme inducers on the soluble enzymes of hepatic phase II biotransformation

Abstract Numerous xenobiotics induce microsomal enzymes such as cytochrome P-450-dependent monooxygenases, epoxide hydrolase, and UDP-glucuronyltransferase by causing an increase in enzyme synthesis. Since induction of soluble enzymes involved in phase II biotransformation has not been thoroughly studied, effects of the following microsomal enzyme inducers on three important soluble enzymes were examined: phenobarbital (PB), 3-methylcholanthrene (3-MC), butylated hydroxyanisole (BHA), isosafrole (ISF), pregnenolone-16α-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and trans-stilbene oxide (TSO). Representative microsomal enzymes of phase I pathways were examined simultaneously to indicate effective induction. The inducers selected produced the expected increases in hepatic cytochrome P-450 (75–170%), ethylmorphine (200–260%), and benzphetamine (100–260%) N-demethylation, benzo[a]pyrene hydroxylation (300%), ethoxyresorufin O-deethylation (2700%), and styrene oxide hydration (100–270%). The soluble conjugative enzymes studied were glutathione S-transferase, N-acetyltransferase, and sulfotransferase. Glutathione conjugation of 1,2-dichloro-4-nitrobenzene, 1-chloro-2,4-dinitrobenzene, and sulfobromophthalein was increased by TSO (100–160%), BHA (60–80%), ISF (60–80%), and PB (60–80%). β-Naphthylamine N-acetyltransferase activity was increased by PCN and 3-MC (60 and 40%, respectively). Only PCN was able to enhance sulfotransferase. Sulfation of 2-naphthol, taurolithocholate, and dehydroepiandrosterone was increased by 28, 111, and 140%, respectively. In conclusion, while microsomal enzymes could be readily induced, activity of soluble phase II enzymes was increased to a much lesser extent. Of the inducers studied, PCN was the most effective at increasing activity of soluble enzymes.

Species variations in biliary excretion of glutathione-related thiols and methylmercury

The biliary excretion of methylmercury is thought to be related to the biliary excretion of nonprotein thiols in rats. Species differences in biliary excretion of glutathione (GSH) and related thiols are unknown; therefore, the relationship between the biliary excretion of GSH-related thiols and methylmercury in five species was studied. The biliary excretion rate of GSH-related thiols and disulfides was 369, 192, 94, 50, and 19 nmol/min/kg for mice, rats, hamsters, guinea pigs, and rabbits, respectively. The main thiol in mouse, hamster, and rat bile was GSH, whereas guinea pig and rabbit bile contained mainly cysteinylglycine (Cys-Gly). The larger percentage of Cys-Gly in guinea pig and rabbit bile was correlated with their greater hepatic gamma-glutamyltranspeptidase (GGT) activity than that observed in the other species. The biliary excretion rate (nmol/min/kg) of methylmercury was approximately 0.8 in mice, rats, and hamsters compared to significantly lower rates in guinea pigs and rabbits (0.15 and 0.03, respectively). It is concluded that the species-specific composition of GSH-related thiols and disulfides in bile is related to species variations in hepatic GGT activity, and that the species variation in biliary excretion of GSH-related thiols does not entirely account for the species variation in methylmercury excretion, indicating other factors are also apparently involved in determining the rate of biliary excretion of methylmercury.

Effect of glutathione depletion on sulfate activation and sulfate ester formation in rats

Sulfation of organic compounds requires activation of inorganic sulfate via formation of adenosine 3-phosphate 5-phosphosulfate (PAPS). Inorganic sulfate can be formed by sulfoxidation of cysteine, which can be derived from GSH. Thus, a decrease in hepatic GSH may impair formation of inorganic sulfate, the synthesis of PAPS, and the sulfation of chemicals. This hypothesis was tested by investigating the effect of GSH depletion on the levels of inorganic sulfate in serum and of PAPS in liver, and on the capacity to form the sulfate conjugate of harmol in rats. Phorone (2 mmol/kg, i.p.) decreased hepatic GSH (97%), serum inorganic sulfate (63%), and hepatic PAPS (48%). Diethyl maleate and vinylidene chloride (6 mmol/kg, each, i.p.) were less effective than phorone in decreasing GSH in liver and inorganic sulfate in serum, and they did not alter hepatic PAPS levels. Three hours after phorone treatment, the nadir of hepatic PAPS concentration, harmol was injected in order to assess sulfation in vivo. After administration of harmol (100 and 300 mumol/kg, i.v.), less harmol sulfate and more harmol glucuronide were found in the serum of phorone-treated rats as compared to control rats. At the higher dosage of harmol, phorone reduced the biliary excretion of harmol sulfate while increasing the biliary excretion of harmol glucuronide. These results indicate that severe GSH depletion decreases PAPS formation and sulfation of chemicals. However, an increase in glucuronidation may compensate for the impaired sulfation.

Simple method for analysis of diquat in biological fluids and tissues by high-performance liquid chromatography☆

A simple HPLC method has been described to quantify diquat in biological fluids and tissues. This method permits separation and quantification of diquat from blood, bile, urine, liver and kidney. It does not require special pretreatment of the samples prior to analysis, nor a specially prepared analytical column. Various concentrations of diquat were added (10-300 nmol/ml or g) to fluids or tissues. Analysis of blank samples revealed no substances that interfere with diquat elution. Excellent recovery (95-105%) was obtained. Diquat (120 mumol/kg, i.v.) was injected to rats and quantified in bile, blood and liver. Concentration of diquat was higher in blood and bile than liver. Therefore, this method is applicable for quantification of diquat in toxicological samples, and may be used to determine structurally similar compounds such as paraquat.

Comparison of biliary excretion of organic anions in mice and rats

Abstract Biliary excretion of cholephilic organic acids in anesthetized, male Swiss-Webster mice was compared to that in male Sprague-Dawley rats. The mouse excreted six of the eight compounds examined at a faster or equal rate than the rat. Indocyanine green, rose bengal, phenol-3,6-dibromsulphthalein disulfonate, and eosine were excreted in mice at a rate 120 to 460% higher than in rats. The excretion rates of bromcresol green and sulfobromophthalein glutathione conjugate were similar in the two species, whereas amaranth was excreted at a slightly lower rate in mice than in rats. Biliary excretion of sulfobromophthalein (BSP), especially its glutathione conjugate, was significantly lower in the mouse which corresponded to a difference in BSP-glutathione transferase activities between the two species (mouse, 0.97; rat, 1.35 μmol/min/g liver). The depression of bile production by cholestatic organic anions was stronger, and the stimulation of bile flow by choleretic acids was weaker in mice than in rats. Differences in biliary bile acid excretion (mouse, 3.62; rat, 1.42 μmol/kg/min), bile flow (mouse, 102; rat, 69 μl/kg/min), and liver weight (mouse, 57; rat, 38 g/kg) but not hepatic ligandin concentration (mouse, 132; rat, 214 nmol BSP/g liver) may explain the variations in the biliary organic anion excretion between mice and rats.

Effect of sulfhydryl-deficient diets on hepatic metallothionein, glutathione, and adenosine 3′-phosphate 5′-phosphosulfate (PAPS) levels in rats☆

Low dietary concentrations of methionine and cysteine are known to decrease hepatic glutathione content. However, it is not known if restricting the dietary content of these sulfur containing amino acids also affects hepatic levels of adenosine 3-phosphate 5-phosphosulfate (PAPS), the cofactor for sulfation, or metallothionein, a protein rich in sulfhydryl groups. Rats were fed diets lacking cysteine and containing various concentrations of methionine (0.15, 0.3, or 0.6%) for 8 days. Control diet contained 0.3% each of methionine and cysteine. Hepatic glutathione levels were decreased approximately 75% in rats fed diets containing 0.15 or 0.3% methionine. In contrast, PAPS and hepatic metallothionein concentrations were not decreased by the low sulfhydryl diets. Additionally, rats on the various diets were challenged by the administration of ZnCl2 (3 mmol/kg. sc). In both control rats and rats maintained on sulfhydryl-deficient diets, ZnCl2 increased hepatic metallothionein to the same level. However, significantly lower levels of PAPS were observed after ZnCl2 in rats receiving sulfhydryl-deficient diets than in controls. In summary, restriction of dietary sulfhydryl markedly decreases the hepatic content of glutathione and has a minor effect on PAPS concentration, but does not decrease the basal hepatic concentration of metallothionein or its induction by ZnCl2.

Inducibility of glutathione S-transferases in hamsters

The effects of 3-methylcholanthrene (3-MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), phenobarbital, trans-stilbene oxide (TSO), pregnenolone-16 alpha-carbonitrile (PCN), dexamethasone, ethanol, isoniazid and butylated hydroxyanisole (BHA) on hepatic glutathione S-transferase (GST) activities toward six substrates were determined in hamsters. TCDD and 3-MC, which are comparatively poor inducers of GSTs in rats, were most effective in enhancing GST activities in hamster liver. In contrast, TSO, BHA and phenobarbital, which are very effective inducers of hepatic GSTs in rats and mice, were ineffective or poor inducers of GSTs in hamster liver. While dexamethasone increased some GST activities, treatments with PCN, ethanol and isoniazid were without effect. The findings indicate that not only the control activity but also the inducibility of hepatic GSTs are different in hamsters from those in other species.