Margaret O. James

Triclosan as A substrate and inhibitor of 3'-phosphoadenosine 5'-phosphosulfate-sulfotransferase and udp-glucuronosyl transferase in human liver fractions

Triclosan is a broad spectrum antibacterial agent used in many household products. Due to its structural similarity to polychlorobiphenylols, which are potent inhibitors of the sulfonation and glucuronidation of 3-hydroxy-benzo[a]pyrene, it was hypothesized that triclosan would inhibit these phase II enzymes. This study was designed to assess the interactions of triclosan as a substrate and inhibitor of 3′-phosphoadenosine 5′-phosphosulfate-sulfotransferases and UDP-glucuronosyltransferases in human liver cytosol and microsomes. Triclosan was sulfonated and glucuronidated in human liver. The apparent Km and Vmax values for triclosan sulfonation were 8.5 μM and 0.096 nmol/min/mg protein, whereas Km and Vmax values for glucuronidation were 107 μM and 0.739 nmol/min/mg protein. Triclosan inhibited the hepatic cytosolic sulfonation of 3-hydroxybenzo(a)pyrene (3-OH-BaP), bisphenol A, p-nitrophenol, and acetaminophen with IC50 concentrations of 2.87, 2.96, 6.45, and 17.8 μM, respectively. Studies of 3-OH-BaP sulfonation by expressed human SULT1A1*1, SULT1A1*2, SULT1B1, and SULT1E1 showed that triclosan inhibited the activities of each of these purified enzymes with IC50 concentrations between 2.09 and 7.5 μM. Triclosan was generally a less potent inhibitor of microsomal glucuronidation. IC50 concentrations for triclosan with 3-OH-BaP, acetaminophen, and bisphenol A as substrates were 4.55, 297, and >200 μM, respectively. Morphine glucuronidation was not inhibited by 50 μM triclosan. The kinetics of 3-OH-BaP sulfonation and glucuronidation were examined in the presence of varying concentrations of triclosan: the inhibition of sulfonation was noncompetitive, whereas that of glucuronidation was competitive. These findings reveal that the commonly used bactericide triclosan is a selective inhibitor of the glucuronidation and sulfonation of phenolic xenobiotics.

The conjugation of phenylacetic acid in man, sub-human primates and some non-primate species

14C-Labelled phenylacetic acid has been administered to man, 14 species of sub-human primates and 11 non-primate species and their urine examined for metabolites. Four amino acid conjugates of this acid have been found in various species, namely, phenacetylglutamine, phenacetylglycine, phenacetyltaurine and diphenacetylornithine, but their occurrence varies with species. In the primates the occurrence of the glutamine and glycine conjugates appears to be correlated with their evolutionary status. Man excretes exclusively the glutamine conjugate, the Old World monkeys the glutamine conjugate and very small amounts of the glycine conjugate, the New World monkeys the glutamine conjugate and significant amounts of the glycine conjugate and the prosimians the glycine conjugate only. The non-primate mammalian species excrete the glycine conjugate and no glutamine conjugate. The two avian species examined also differed, since the pigeon excreted the glycine conjugate, whereas the domestic hen excreted mainly the ornithine conjugate with small amounts of the glycine conjugate. The conjugation of phenylacetic acid with taurine is reported for the first time. It occurs in all the species examined except the vampire bat and domestic hen, but its quantitative occurrence is haphazard amongst the species examined. It was found in significant amounts in the pigeon, ferret, bushbaby, capuchin monkey, squirrel monkey, mona monkey and baboon, but in minor amounts in other species.

Hepatic and extrahepatic metabolism, in vitro, of an epoxide (8-14C-styrene oxide) in the rabbit

Abstract Two epoxide-metabolizing enzymes, glutathione- S -epoxide transferase and epoxide hydrase, were studied in subcellular fractions of rabbit liver, lungs, intestinal mucosa and kidney. Glutathione- S -epoxide transferase in soluble fraction was assayed by a new specific radiochemical method using styrene oxide (−8- 14 C) as substrate, and some properties of this enzyme are described. Liver had the highest specific activity of each enzyme, but there was no correlation between the relative specific activities of glutathione- S -epoxide transferase and epoxide hydrase in the extrahepatic organs. Feeding rabbits a purified diet did not alter the specific activities of epoxide hydrase or glutathione- S -epoxide transferase in liver and intestine. Rat and guinea pig had much higher specific activities of glutathione- S -epoxide transferase than rabbit in liver and kidney, and slightly higher specific activities in lung and intestine. Species did not differ markedly in epoxide hydrase activities when the same organ (liver, lung, intestine or kidney) was compared in rabbits, rats and guinea pigs, except that rat intestine had much lower epoxide hydrase activity than intestine from rabbit or guinea pig.

Cytochrome P450 monooxygenases in crustaceans

1. The hepatopancreas is the major site of cytochrome P450-dependent xenobiotic monooxygenation in crustacean species, but the presence of monooxygenase inhibitors in hepatopancreas microsomes and cytosol from many decapod species has impeded in vitro studies. Cytochrome P450 and monooxygenase activities have been reported in other crustacean organs including the antennal gland (green gland) and stomach. 2. NADPH cytochrome c reductase activity is often very low (typically less than 10 nmol cytochrome c reduced/min per mg microsomal protein) in hepatopancreas microsomes from crustacean species. NADPH cytochrome P450 reductase activity has not yet been detected in crustacean hepatopancreas microsomes. 3. The cytochrome P450 present in hepatopancreas of several crab species and the spiny lobster has been resolved into several fractions by chromatography on DEAE-cellulose. One form of cytochrome P450 from spiny lobster has been purified to 12 +/- 2 nmol/mg protein. 4. Reconstitution studies with spiny lobster hepatopancreas P450 have shown that the vertebrate sex steroids, progesterone and testosterone, are excellent substrates, whereas ecdysone--the crustacean molting hormone--is not a substrate. Activity was found with several xenobiotic substrates including benzphetamine, aminopyrine, benzo(a)pyrene, ethyl-, benzyl- and pentyl-phenoxazones and ethoxycoumarin. Highest activities (greater than 50 nmol/min per nmol P450) were found for N-demethylation of benzphetamine and aminopyrine. 5. The ability of agents which induce vertebrate cytochrome P450 to induce cytochrome P450 in crustaceans is still unclear. Some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type. Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity.

CYTOCHROMES P450 IN CRUSTACEA

Since the last review of this topic, further insight has been gained into the presence and functions of cytochrome P450 proteins in the hepatopancreas and other organs of aquatic crustacean species, although progress has been slow relative to the advances in other species. Recent studies with several lobster, shrimp, crab and crayfish species suggest that cytochromes P450 in the 2 and 3 families are the most abundant forms in hepatopancreas microsomes. Substrates normally metabolized by CYP2 and CYP3 family members are monooxygenated more rapidly by crustacea than substrates normally metabolized by CYP1 family enzymes, e.g. erythromycin, testosterone and aminopyrine are much more rapidly monooxygenated than ethoxyresorufin. Some progress has been made in cloning and sequencing crustacean P450 forms. CYP2L1 and CYP2L2 cDNA sequences have been cloned from spiny lobster hepatopancreas libraries, and there was evidence for at least two more cytochromes P450 in spiny lobster hepatopancreas. An area of continued interest, but of no consensus or general findings, relates to the presence and inducibility of CYP1 family members in crustacea. Some studies indicate weak induction of total cytochrome P450 and increased turnover of substrates normally associated with CYP1, while others show no effect of the classic inducers that act at the Ah receptor in vertebrates. A few studies of the roles of cytochromes P450 in the biosynthesis and degradation of steroids, including ecdysteroids, have been published. Further studies are needed to understand the regulation and normal function of the crustacean cytochromes P450.

Hepatic microsomal mixed-function oxidase activities in several marine species common to coastal Florida

1. NADPH-dependent hepatic microsomal mixed-function oxidase activities were measured in a number of marine vertebrate and invertebrate species common to the Florida Atlantic coast, using benzo(a)pyrene, 7-ethoxycoumarin, benzphetamine and aniline as substrates. Optimal assay conditions were established using sheepshead hepatic microsomes. 2. Mixed-function oxidase activity was easily detected in hepatic microsomes from teleost and elasmobranch fish, but was low or undetectable in hepatopancreas microsomes from crustaceans. However, the cytochrome P-450 content of hepatopancreas microsomes often exceeded that found in microsomes from teleost or elasmobranch liver. 3. Mixed-function oxidase activity in microsomes prepared from extrahepatic organs was usually much lower than was found in hepatic microsomes. However, for two teleost species (sheepshead and black drum), benzphetamine N-demethylase activity of gill microsomes approached that found in hepatic microsomes. 4. The temperature optimum for in vitro mixed-function oxidase activity was higher in the Florida fish tested than has been reported for cold-water acclimated marine or freshwater fish.

Epoxide hydrase and glutathione S-transferase activities with selected alkene and arene oxides in several marine species

Epoxide hydrase and glutathione (GSH) S-transferase activities were measured in subcellular fractions prepared from liver or hepatopancreas and some extrahepatic organs of a number of marine species common to Maine or Florida. These activities were easily detected in the species studied. In fish, hepatic GSH S-transferase activities were normally higher than hepatic epoxide hydrase activities for the alkene oxide (styrene oxide and octene oxide) and arene oxide (benzo[a]pyrene 4,5-oxide) substrates studied, whereas in crustacea, hepatopancreas epoxide hydrase activities were higher than hepatopancreas GSH S-transferase activities with the same substrates. Extrahepatic organs from fish and crustacea usually had higher GSH S-transferase activities than epoxide hydrase activities with the alkene and arene oxide substrates. GSH S-transferase activity was also found in liver or hepatopancreas of every aquatic species studied and in a number of extrahepatic organs, when 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene served as substrate.

The state of in vitro science for use in bioaccumulation assessments for fish

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Programs Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

Interactions of cytosolic sulfotransferases with xenobiotics.

Abstract Cytosolic sulfotransferases are a superfamily of enzymes that catalyze the transfer of the sulfonic group from 3′-phosphoadenosine-5′-phosphosulfate to hydroxy or amine groups in substrate molecules. The human cytosolic sulfotransferases that have been most studied, namely SULT1A1, SULT1A3, SULT1B1, SULT1E1 and SULT2A1, are expressed in different tissues of the body, including liver, intestine, adrenal, brain and skin. These sulfotransferases play important roles in the sulfonation of endogenous molecules such as steroid hormones and neurotransmitters, and in the elimination of xenobiotic molecules such as drugs, environmental chemicals and natural products. There is often overlapping substrate selectivity among the sulfotransferases, although one isoform may exhibit greater enzyme efficiency than other isoforms. Similarly, inhibitors or enhancers of one isoform often affect other isoforms, but typically with different potency. This means that if the activity of one form of sulfotransferase is altered (either inhibited or enhanced) by the presence of a xenobiotic, the sulfonation of endogenous and xenobiotic substrates for other isoforms may well be affected. There are more examples of inhibitors than enhancers of sulfonation. Modulators of sulfotransferase enzymes include natural products ingested as part of the human diet as well as environmental chemicals and drugs. This review will discuss recent work on such interactions.

Effect of 3-methylcholanthrene on monooxygenase, epoxide hydrolase, and glutathione S-transferase activities in small estuarine and freshwater fish

Abstract Monooxygenase activities, epoxide hydrolase, and glutathione S -transferase activities were measured in fractions prepared from pooled livers of three small estuarine fish species, Fundulus grandis, Cyprinodon variegatus , and Poecilia latipinna , and three freshwater species, Oryzias latipes, Peocilia reticulata , and Pimephales promelas . Interspecies differences in xenobiotic metabolizing activities of up to 10-fold were observed, with the smaller freshwater species generally having lower activities than the estuarine species. Groups of each species were injected with microliter volumes of corn oil or a solution of 3-methylcholanthrene (3MC) in corn oil, and enzyme activities measured 4–5 days later in pooled liver fractions from control or 3MC-treated fish. Although statistical analysis of induction effects was not possible, monooxygenase activities with ethoxyresorufin and benzo( a )pyrene were 2.5–15 times higher in 3MC-treated fish than in corn oil or untreated control groups. In 3MC-treated C. variegatus and F. grandis , microsomal cytochrome P-450 content was doubled and the spectral maximum of the CO-reduced microsomes was shifted from 449.6 to 448.5 nm. Microsomes from 3MC-treated C. variegatus and F. grandis metabolized benzo( a )pyrene 5–6 times faster than controls (per mg protein), but the proportion of 9,10- and 7,8-dihydrodiol metabolites was lower in 3MC-treated fish, suggesting that epoxide hydrolase may be rate limiting for production of dihydrodiols. 3MC treatment did not alter styrene oxide hydrolase or glutathione S -transferase activities at the time point tested.