Hepatic AHH Research Articles

Induction of CYP1A by a diterpene quinone tanshinone IIA isolated from a medicinal herb Salvia miltiorrhiza in C57BL/6J but not in DBA/2J mice

Effects of tanshinone IIA, an active diterpene quinone of the herbal medicine Salvia miltiorrhiza (Danshen), on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in the arylhydrocarbon (Ah)-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice. Oral treatment of tanshinone IIA caused a dose-dependent increase of liver microsomal 7-methoxyresorufin O-demethylation (MROD) activity in B6 but not in D2 mice. In B6 mice, tanshinone IIA increased hepatic benzo(a)pyrene hydroxylation (AHH), 7-ethoxyresorufin O-deethylation, MROD, and 7-ethoxycoumarin O-deethylation activities. The levels of Cyp1A2 protein and mRNA were elevated. On the contrary, in D2 mice, tanshinone IIA decreased hepatic AHH and nifedipine oxidation activities and the CYP3A protein level without affecting other activities determined. Cyp1A2 protein and mRNA levels were not affected by tanshinone IIA in D2 mice. Tanshinone IIA had no effects on UGT and GST activities in both B6 and D2 mice. These results demonstrated that induction of CYP1A2 by tanshinone IIA depended on the Ah-responsiveness and occurred at pre-translational level.

Protective effects of baicalein and wogonin against benzo[ a]pyrene- and aflatoxin B 1-induced genotoxicities

To evaluate the protective effects of baicalein and wogonin against benzo[ a]pyrene- and aflatoxin (AF) B 1-induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo[ a]pyrene and AFB 1 were studied in C57BL/6J mice. Baicalein and wogonin reduced benzo[ a]pyrene and AFB 1 genotoxicities as monitored by the umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. Baicalein added in vitro decreased liver microsomal benzo[ a]pyrene hydroxylation (AHH) activity with an ic 50 of 33.9 ± 1.4 μM at 100 μM benzo[ a]pyrene. Baicalein also inhibited AFQ 1 and AFB 1-epoxide formation from AFB 1 (50 μM) oxidation (AFO) with ic 50 values of 22.8 ± 1.4 and 5.3 ± 0.8 μM, respectively. However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 μM wogonin was only about 51–65%. Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively. Baicalein treatment resulted in 39 and 32% decreases in AFQ 1 and AFB 1-epoxide formation from liver microsomal AFO, respectively. Wogonin treatment resulted in 39 and 47% decreases in AFQ 1 and AFB 1-epoxide formation, respectively. A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo[ a]pyrene via gastrogavage. These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo[ a]pyrene- and AFB 1-induced hepatic toxicities and that wogonin had a stronger protective effect in vivo.

Effects of baicalein and wogonin on drug-metabolizing enzymes in C57BL/6J mice

Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.

Polychlorinated diphenylethers: origin, analysis, distribution, and toxicity in the marine environment.

PCDEs are present as impurities in chlorophenol preparations, which are often used as wood preservatives. Other sources of PCDEs such as municipal waste incinerators seem to be of minor importance. It is estimated that, worldwide 250-2500 metric tons of PCDEs have been produced, which is a lesser amount compared to the production figures of polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs), or polychlorinated naphthalenes (PCNs). The required analytical methods are rather complex and are comparable to methods used for polychlorinated dibenzo-p-dioxins (PCDDs) and furans (PCDFs). Gas chromatography with low resolution or high resolution mass spectrometry or with electron-capture detection is recommended for the final determination of PCDEs. There is only limited information on the presence of PCDEs in the marine environment. High concentrations of PCDEs were found in Baltic white-tailed sea eagles (up to 13 mg/kg lipid weight per congener). Total PCDE concentrations of 50-300 micrograms/kg were reported in cod liver oil samples. PCDEs are immunotoxic and show a clear potency as inducers of hepatic microsomal AHH and EROD activity. Although PCDE concentrations in marine organisms are expected to be lower than those of PCBs, PBDEs, and PCNs, because of their toxic properties, persistence, and hydrophobic and bioaccumulative character and because of the limited information on these concentrations in marine organisms, further research on these concentrations in marine organisms is recommended.

Modulation by ascorbic acid of the cutaneous and hepatic biochemical effects induced by topically applied benzanthrone in mice

Modulation of biochemical markers by ascorbic acid was investigated in mice to which benzanthrone (BA) was applied topically (150 nmol/mouse) twice a week for 34 wk. After BA exposure without ascorbic acid, in the skin there were significant decreases in the activities of aryl hydrocarbon hydroxylase (AHH; 38% decrease relative to controls) and ethoxyresorufin- O-deethylase (EROD; 39%), and enhancement of the activities of quinone reductase (41% increase), tyrosinase (82%) and histidine decarboxylase (HDC; 190%). BA exposure also caused significant inhibition of hepatic AHH, EROD and glutathione- S-transferase activities, with concomitant increases in the activities of histidase (52%) and HDC (58%). Ascorbic acid given orally (5 mg/mouse) or topically (1 mg/mouse) twice weekly for 34 wk to BA-treated mice resulted in substantial protection against the effects of BA on these enzyme markers in both the skin and the liver. These results suggest that ascorbic acid could be useful in preventing the biochemical and toxicological manifestations caused by BA in laboratory animals.

Potentiation and Antagonism of 2,3,7,8-Tetrachlorodibenzo- p-dioxin Effects in a Complex Environmental Mixture

There is increasing need to understand the toxicity of complex environmental mixtures. The organic phase of a leachate (OPL) from the Love Canal chemical dump site is a complex mixture that contains over 100 organic compounds, including 0.74 ppm 2,3,7,8,-tetrachlorodibenzo- p-dioxin (TCDD). Mice congenic at the Ah locus were used to evaluate several toxic effects of the OPL, including immune function and hepatic enzyme induction. OPL toxicity was compared with that of pure TCDD in both C57BL/6J Ah b/b and congenic C57BL/6 Ah d/d (B6.D2) mice. Mice were given single oral doses of up to 2 g OPL/kg or 100 μg TCDD/kg, immunized, and evaluated after 7 days. The TCDD equivalent of the OPL was determined to be 3.9 and 5.0 ppm in C57BL/6J and B6.D2 mice, respectively. This is six times the TCDD content. The Ah phenotype-dependent response ratio was calculated by dividing the dose required to cause an effect in the B6.D2 strain by the dose causing the same effect in the C57BL/6J strain. Ratios based on both ED5Os and the lowest observed adverse effect levels were used to determine whether each adverse effect was Ah phenotype-dependent, the extent to which TCDD contributed to the effect, whether there were interactive effects between the AhR ligands and nonligands and if they were additive, antagonistic, or synergistic, and whether the response was predictable based on the known chemical composition of the mixture. It was concluded that the non-TCDD component potentiated TCDD immune suppression, and possibly thymic atrophy, through AhR mechanisms. In contrast, this analysis indicated that the non-TCDD component of the OPL antagonized the ability of the TCDD component to induce hepatic AHH activity whereas OPL hepatomegaly was caused primarily by the non-TCDD component of the OPL. This study demonstrates that the toxicity of mixtures containing TCDD may not be accurately predicted based on the TCDD content alone and that this approach could be useful in the toxicologic assessment and management of environmental contamination.

Differential expression and regulation of members of the cytochrome P450IA gene subfamily in human tissues

Cigarette smoking increases phenacetin O-deethylase (POD) activity in both the liver and placenta in man, but aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity is increased only in the placenta. Whilst there was no correlation between hepatic POD and AHH activities (rs = 0.42, P greater than 0.1), there was a highly significant correlation between these two activities in placenta (rs = 0.76, P less than 0.02). On Western blotting of human liver samples with an antibody specific to cytochrome P450IA2 in the rat, only the orthologue of P450IA2 could be detected. This antibody inhibited greater than 70% of hepatic high-affinity POD activity but had no effect on the placental activity. Furafylline, a methylxanthine that acts as a highly specific inhibitor of P450IA2-dependent activities in man, inhibited all of the high-affinity component of POD activity in human liver, but was at least three orders of magnitude less potent an inhibitor of placental POD and of both hepatic and placental AHH activities. As previously shown in the rat, exposure of man to polycyclic aromatic hydrocarbons, present in cigarette smoke, differentially induces P450IA2 in the liver and P450IA1 in extrahepatic tissues, at least in the placenta. Again, as in the rat, POD activity in the liver is catalysed by P450IA2, but in the placenta of women exposed to polycyclic aromatic hydrocarbons in cigarette smoke POD activity is catalysed by another isoenzyme, most likely P450IA1. Thus, tissue-dependent induction and substrate specificity of members of the P450IA family in man, at least in the placenta, appear to be the same as previously shown in the rat.

Synergistic interactions of 2, 3, 7, 8-TCDD and 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl in C57BL/6J and DBA/2J mice: Role of the Ah receptor

Treatment of C57BL/2J mice with 2, 2′, 4, 4′, 5, 5′-hexachlorobiphenyl (HCBP, 500 μmol/kg) elevated hepatic cytosolic Ah receptor levels 82–107% for up to 14 days. Scatchard analysis of the [ 3H] 2, 3, 7, 8-TCDD (TCDD)-Ah receptor saturation binding curves from corn oil and HCBP treated rats gave K D values of 0.80 and 0.90 nM, respectively and confirmed that treatment with HCPB did not significantly alter receptor-radioligand affinities. Administration of HCBP to DBA/2J mice did not result in detectable hepatic cytosolic Ah receptor levels. Cotreatment of C57BL/6J mice with HCBP (500 μmol/kg) at a dose level of TCDD (1 nmol/kg) which elicite less that 10% of the maximum inductions response resulted in significant synergistic iduction of hepatic EROD and AHH [compared to animals treated only with TCDD (1 nmol/kg)]. In contrast, contreatment of C57BL/6J mice with HCBP (500 μmol/kpg) and maximally inducing dose levels of TCDD (100 or 500 nmol/kg) resulted in either a slight or no difference in the induction of AHH or EROD compared to the induction responses observed in mice treated only with TCDD. In contrast, contreatment of DBA/2J mice with TCDD and HCBP (500/gmmol/kg) resulted in significant synergistic induction of AHH and EROD at both submaximal (10–50 nmol/kg) and maximal (5000 nmol/kg) induction levels of TCDD. The only significant interactive effect of HCBP (500 μmol/levels of TCDD. The only significant interactive effect of HCBP (500 μmol/kg) on the toxicity of TCDD in C57BL/6J and DBA/2J was protection from body weight loss observed after cotreatment of HCBP and TCDD in DBA/2J mice

The effect of intraovarian injection of benzo( a)pyrene on primordial oocyte number and ovarian aryl hydrocarbon [benzo( a)pyrene] hydroxylase activity

The effect of intraovarian (io) injection of benzo(a)pyrene (BP) on primordial oocyte number, ovarian aryl hydrocarbon hydroxylase (AHH, EC 1.14.14.1) activity, and hepatic AHH activity and P-450 content was investigated in C57BL/6N (B6) and DBA/2N (D2) mice. Ovaries were exteriorized through dorsolumbar incisions under ether anesthesia, and 1.0 μl of corn oil or corn oil containing BP was injected directly into the ovary. Bilateral io injection of BP was ovotoxic in a time and dose dependent manner. Primordial oocyte destruction was maximal 8 days after injection in both B6 and D2 mice. Unilateral io injection of BP destroyed oocytes only in the treated ovary. The threshold dose for primordial oocyte destruction was similar in both strains, approximately 50 ng/ovary. However, ED50's were different; 1.1 μg/ovary in B6 and 8.9 μg/ovary in D2 mice. Intraperitoneal treatment with α-naphthoflavone (80 mg/kg) inhibited the oocyte destruction by io treatment with BP (10 μg/ovary) in both B6 and D2 mice. In B6 mice, ovarian AHH activity was induced by BP in the treated ovary after either bilateral, or unilateral io treatment. However, io BP treatment did not change ovarian AHH activity in D2 mice. Hepatic AHH activity and P-450 content was not altered by io BP treatment in either mouse strain. These results strengthen the hypothesis that the ovary has the capability of metabolizing BP to ovotoxic products.

Xenobiotics in Gametes of Lake Michigan Lake Trout (Salvelinus namaycush) Induce Hepatic Monooxygenase Activity in Their Offspring

Xenobiotics in Gametes of Lake Michigan Lake Trout (Salvelinus namaycush) Induce Hepatic Monooxygenase Activity in Their Offspring. BINDER, R. L., AND LECH, J. J. (1984). Fundam. Appl. Toxicol. 4, 1042–1054. Eggs spawned from Lake Michigan lake trout contain a number of xcnobiotic compounds, including polychlorinated biphenyls (PCBs). To assess whether this contamination is sufficient to induce hepatic cytochrome P-450-dependent monooxygenase (MO) activity during early development, the hepatic MO systems of laboratory-cultured offspring of Lake Michigan, Green Bay, and Marquette Hatchery lake trout were compared. Additionally, the induction of hepatic cytochrome P-450 systems in developing lake trout by the commercial PCB mixture, Aroclor 1254 (A1254), was characterized. During late embryonic development and at the swim-up stage, the hepatic MO systems of the feral lake trout offspring appeared induced, based on levels of aryl hydrocarbon bydroxytase (AHH) activity that were 3.5- to 8.6-fold higher than the hatchery control levels. Furthermore, at the swim-up stage the feral trout offspring resembled A1254-treated hatchery fry with regard to the degree of inhibition of hepatic AHH activity by α-naphthoflavone, and the presence of an inducible Mr = 58,000 polypeptide in hepatic microsomes. The levels of aminopyrine N-demethylase activity, which was relatively unresponsive to inducers, were moderately lower in the Lake Michigan and Green Bay swim-up fry compared to the hatchery control levels. After 7 months of posthatching laboratory culture, when residues of xenobiotics present at fertilization were greatly diluted by growth, the hepatic MO systems of the Lake Michigan and hatchery trout offspring appeared essentially indistinguishable with regard to a number of parameters. These results indicate that the differences observed between the hatchery and feral trout offspring at earlier stages were not likely to have a genetic basis, but rather were due to a xenobiotic-type induction. Dose-response experiments and residue analysis data indicated that residues of commercial PCB mixtures were at least partially responsible for the effects described here. Overall, these data provide very strong evidence that contamination of Lake Michigan lake trout gametes by PCBs and possibly other xenobiotics caused induction of hepatic MO activity in their progeny. Fish embryos and fry with induced MO systems may have an increased sensitivity to the toxicity of certain environmental contaminants that are metabolically activated by cytochrome P-450.

Biochemical Evidence that Winter Flounder (Pseudopleuronectes americanus) have Induced Hepatic Cytochrome P-450-Dependent Monooxygenase Activities

Livers from winter flounder (Pseudopleuronectes americanus) captured near Mount Desert Island, Maine, showed marked variation in hepatic benzo[a]pyrene hydroxylase (AHH; from 0.04 to 8.8 FU∙min−1∙mg protein−1) and 7-ethoxyresorufin deethylase activities (7-ERD; from < 2 to 1165 pmol∙min−1∙mg protein−1), and a dichotomy in the effect of 7,8-benzoflavone (ANF) added in vitro on AHH activity. Based on this ANF effect, the flounder could be divided into two groups. One group had high 7-ERD activity and high AHH activity which was inhibited by ANF; the other group had low 7-ERD activity and low AHH activity which was enhanced by ANF. Sex, weight, length, liver weight, gonad weight/body weight ratio, and liver/body weight ratio explained only a small part of the variability in hepatic AHH activities. Electrophoretograms of hepatic microsomes from flounder treated with 1,2,3,4-dibenzanthracene (DBA) or 5,6-benzoflavone (βNF) showed a novel or enriched polypeptide species present near 57 000 daltons, in the molecular weight (MW) range of known cytochrome P-450 isozymes. A polypeptide of similar MW was only faintly discernable in liver microsomes from untreated flounder whose hepatic AHH activity was much lower than that of the treated flounder, whereas a band of this MW was prominent in hepatic microsomes from untreated flounder with high hepatic AHH activity. These results suggest that many of the winter flounder captured near Mount Desert Island, Maine have induced hepatic monooxygenase activity due to exposure to PAH or PAH-like inducers present in their natural habitat.

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[ a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.

Monooxygenase activity of human liver in microsomal fractions of needle biopsy specimens.

1 Methods are described for the determination of mixed function oxidase activity in microsomal fractions from percutaneous needle biopsies of human liver. 2 Activities of needle biopsy samples were comparable with those of wedge biopsy sample obtained at laparotomy from different subjects. 3 Although cytochrome P-450 content of liver from rat and man was similar, human AHH activity was only 10% of that in the rat. 4 In biopsies with preserved hepatic architecture, AHH activity and cytochrome P-450 content showed a significant positive correlation. 5 Cigarette smoking significantly increased both AHH activity and turnover number, but not cytochrome P-450 content, of biopsies with normal architecture. 6 The presence of liver disease caused a significant decrease in AHH activity and cytochrome P-450 content.

Liver and mammary arylhydrocarbon hydroxylase and epoxide hydratase in lactating rats fed polybrominated biphenyls

Female rats were fed polybrominated biphenyls (PBBs) (50 ppm) from day 8 of gestation through day 14 postpartum. Hepatic and mammary liver to body weight ratios, microsomal protein, arylhydrocarbon (benzo(a)pyrene) hydroxylase (AHH) activity and epoxide hydratase (EH) activities were measured. Exposure to PBBs significantly increased liver to body weight ratio, hepatic microsomal protein and hepatic AHH and EH activities. Mammary AHH activity was increased and EH activity was decreased after PBBs. These data demonstrate that AHH and EH are present in mammary tissue and can be altered by exposure to PBBs.

Induction of hepatic aryl hydrocarbon hydroxylase in salmon exposed to petroleum dissolved in seawater and to petroleum and polychlorinated biphenyls, separate and together, in food

1. Hepatic AHH is induced in coho salmon exposed to 1 ppm PCB's in the diet; however, comparable experiments with 1 ppm of Prudhoe Bay crude oil did not significantly alter the AHH activities. These findings suggest that in aquatic food-chains containing both petroleum hydrocarbons and PCB's, the latter compounds may exert a predominant influence on the induction of the hepatic AHH enzyme system. 2. Despite the lack of induction of hepatic AHH with 1 ppm Prudhoe Bay crude oil in the diet, young coho salmon exposed to 150 ppb (0.15 ppm) of a seawater-soluble fraction for 6 days exhibited a significant induction of this enzyme system. Thus, crude oil components of relatively high water solubility (e.g., methylated naphthalenes and polyalkylated benzenes) may be more effective than high molecular weight insoluble components in inducing AHH.