Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Mouse skin tumor-initiating activity of 5-, 7-, and 12-methyl- and fluorine-substituted benz[a]anthracenes.

As an approach for elucidation of structure activity relationships that underlie the exceptionally large difference in carcinogenic activity between benz[a]anthracene and 7,12-dimethylbenz[a]anthracene (7,12-DMBA), 11 methyl- and/or fluorine-substituted benz[a]anthracenes were evaluated for tumor-initiating activity on mouse skin. Outbred CD-1 and outbred Sencar mice received a single topical application of the hydrocarbons followed by twice weekly applications of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate for 16-26 weeks. 7,12-DMBA was almost two orders of magnitude more active as a tumor-initiator than 7- and 12-methylbenz[a]anthracene. Methyl substitution at the 7- and 7,12-positions of benz[a]anthracene was significantly more effective in the enhancement of tumorigenic activity than fluorine substitution at these positions. Although 7-fluorobenz[a]anthracene, 12-fluorobenz[a]anthracene, and 7,12-difluorobenz[a]anthracene had only 0.15, 0.26, and less than 0.005 times the tumor-initiating activity of their respective methyl-substituted derivatives, they were severalfold more active than benz[a]anthracene. 7-Fluorobenz[a]anthracene was slightly less active than 12-fluorobenz[a]anthracene, whereas 7-methylbenz[a]anthracene was about twofold more than 12-methylbenz[a]anthracene. For 7,12-di-substituted benz[a]anthracenes, 7-methyl-12-fluorobenz[a]anthracene was more than twice as tumorigenic as 7-fluoro-12-methylbenz[a]anthracene, but each was individually more active than 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene, respectively. Both fluorinated compounds were much less active than 7,12-DMBA. Substitution of fluorine or methyl at the 5-position of 7-methylbenz[a]anthracene and substitution of fluorine at the 5-position of 12-methylbenz[a]anthracene dramatically reduced their tumorigenic activity.

Fluorine substitution as a probe for the role of the 6-position of benzo[a]pyrene in carcinogenesis.

The tumorigenic activities of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-F-BP) were compared to determine whether an unsubstituted 6-position is important for the carcinogenic effect of BP. Highly purified samples of 6-F-BP and BP had similar activities for the induction of lung adenomas in Swiss Webster mice treated before weaning. The 6-fluoro derivative, however, had about one-half as much activity as BP for the initiation of skin papillomas in CD-1 mice. Similarly, 6-F-BP (approximately equal to 90% purity) had about one-half the activity of BP for the induction of skin tumors in C57BL/6J mice given repetitive treatments of the hydrocarbons and for the induction of sarcomas in C3H/fCum mice given a single sc injection. 6-F-BP (approximately equal to 90% purity) had activity similar to that of BP for induction of sarcomas at the sc injection site in Fischer 344 rats. These results and related data indicate the need for detailed metabolic studies whenever fluorine substitution is used as a probe to assess the role of the unsubstituted position in the carcinogenicity of the parent compound.

Purified form of cytochrome P-450 from rainbow trout with high activity toward conversion of aflatoxin B1 to aflatoxin B1-2,3-epoxide.

Aflatoxin B1, the most potent hepatic chemical carcinogen known, is activated to the putative product aflatoxin B1-2,3-epoxide via a cytochrome P-450-dependent reaction. Mt. Shasta rainbow trout is the most sensitive species known to the hepatocarcinogenic effects of aflatoxin B1. We have previously isolated and purified a minor form of cytochrome P-450 from this strain of rainbow trout, with a lambda max in the carbon monoxide-reduced difference spectrum of 449.5 nm and a molecular weight of 54,000. In this study, we have compared in a reconstituted system this trout P-450 to trout cytochrome P-448 and rat cytochrome P-450 and P-448 for metabolism and activation of aflatoxin B1. Trout cytochrome P-450 had much higher activity towards aflatoxin B1 and a greater degree of regioselectivity in the formation of aflatoxin B1-2,3-dihydroxy-2,3-dihydrodiol and was much more efficient in producing aflatoxin B1 covalent adducts with DNA. The existence of such a form of cytochrome P-450 in Mt. Shasta rainbow trout may be responsible for the acute sensitivity of this strain to the carcinogenic effects of aflatoxin B1.

Metabolism and tumorigenicity of 7-, 8-, 9-, and 10-fluorobenzo(a)pyrenes.

The skin tumor-initiating activities of 7-, 8-, 9-, and 10-fluorobenzo(a)pyrenes have been compared to that of benzo(a)pyrene in female Sencar mice after 16 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate. Single initiating doses of 200 or 400 nmol of each hydrocarbon were tested, and the mice were treated twice weekly with 3.2 nmol of the promoter. Under these conditions, benzo(a)pyrene caused an average of 2.9 and 5.7 papillomas/mouse, respectively, whereas none of the four fluorinated hydrocarbons had significant tumor-initiating activity. Examination of the hepatic metabolism of 7- and 8-fluorobenzo(a)pyrene revealed that a 7,8-dihydrodiol was not detected as a metabolite; thus, the bay-region diol-epoxide pathway known to be responsible for the tumorigenic activity of benzo(a)pyrene is blocked. Although 7,8-dihydrodiols are formed from 9- and 10-fluorobenzo(a)pyrene, these dihydrodiols with fluorine substituted on the 9,10-double bond may not be converted to diol-epoxides by the cytochrome P-450 system, or such fluorinated 7,8-diol-9,10-epoxides may not be tumorigenic.

Purification of cytochromes P-448 from beta-naphthoflavone-treated rainbow trout.

Rainbow trout were treated with beta-naphthoflavone and the hepatic microsomal cytochrome P-450 solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Chromatography on tryptamine-Sepharose 4B gave a single cytochrome P-450 peak which was further resolved into three components by elution from DEAE-Sepharose. The two main peaks were then chromatographed on hydroxyapatite and a total of four fractions obtained. Two of these fractions had similar properties and significantly metabolized [14C]benzo[a]pyrene in a reconstituted system containing rat cytochrome P-450 reductase. This activity was inhibited by alpha-naphthoflavone but not by metyrapone of SKF-525A. Purified cytochromes P-448 from 3-methylcholanthrene-treated rat had similar spectral properties and activity towards [14C]benzo[a]pyrene suggesting similarities between these forms.

Immunological characterization of flavin-containing monooxygenases from the liver of rainbow trout (Oncorhynchus mykiss): sexual- and age-dependent differences and the effect of trimethylamine on enzyme regulation.

Polyclonal antibodies raised against flavin-containing monooxygenase (FMO) enzymes purified from pig liver and rabbit lung were used in conjunction with N,N-dimethylaniline (DMA) N-oxidase to better characterize FMO from the liver of rainbow trout (Oncorhynchus mykiss). Two proteins reacted with polyclonal antibodies raised against pig liver FMO (PL-1 and PL-2) and anti-rabbit lung FMO (RL-1 and RL-2). Although there was no difference in DMA N-oxidase observed between sexually mature male and female trout liver microsomes, RL-2 and PL-2 were significantly less than RL-1 and PL-1, respectively, in sexually mature females. FMO activity and protein content increased as fish aged. DMA oxidase and FMO isozymes were unaltered after pretreatment with the endogenous substrate trimethylamine. Since antibodies to the purified mammalian enzymes react with proteins of similar MW in trout, some forms of FMO appear to be structurally conserved through evolution.

Immunological characterization of constitutive isozymes of cytochrome P-450 from rainbow trout. Evidence for homology with phenobarbital-induced rat P-450s.

Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.

In vitro glucuronidation of xanthohumol, a flavonoid in hop and beer, by rat and human liver microsomes.

Xanthohumol (XN) is the major prenylated flavonoid of hop plants and has been detected in beer. Previous studies suggest a variety of potential cancer chemopreventive effects for XN, but there is no information on its metabolism. The aim of this study was to investigate in vitro glucuronidation of XN by rat and human liver microsomes. Using high-performance liquid chromatography, two major glucuronides of XN were found with either rat or human liver microsomes. Release of the aglycone by enzymatic hydrolysis with beta-glucuronidase followed by liquid chromatography/mass spectrometry and nuclear magnetic resonance analysis revealed that these were C-4' and C-4 monoglucuronides of XN.

Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells.

The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR.

Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from beta-naphthoflavone-fed rainbow trout.

We have purified five isozymes of liver microsomal (LM) P-450 from beta-naphthoflavone-fed rainbow trout. Four forms (LM3, LM1, LM4a and LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMx into two components, LM2 and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a lambda max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4b by Mr, lambda max, or activity towards benzo[a]pyrene. Form LM2 (2% yield) had a specific content of 10.8, a Mr of 54,000, a lambda max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, LM1 and LM3. Neither LM1 nor LM3 was active towards benzo[a]pyrene. The properties of LM2, LM4a and LM4b were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4a and LM4b each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4a or LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4a and LM4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to alpha-naphthoflavone, and regioselectivity towards benzo[a]pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.

Pyrrolizidine alkaloid-induced alterations in benzo[alpha]pyrene metabolism and binding of benzo[alpha]pyrene metabolites to deoxyribonucleic acid.

Pretreatment of rats by oral administration of jacobine, a pyrrolizidine alkaloid and inducer of epoxide hydrolase, produced a marked shift in hepatic microsomal metabolism in vitro of benzo[alpha]pyrene. The formation of 9-hydroxybenzo[alpha]pyrene and 7,8-dihydroxy-7,8-dihydrobenzo[alpha]pyrene was decreased whereas the formation of 4,5-dihydroxy-4,5-dihydrobenzo[alpha]pyrene was increased following jacobine treatment. This shift in the ratio of benzo[alpha]pyrene metabolites was accompanied by a significant reduction in DNA binding. Addition of purified epoxide hydrolase to control or jacobine microsomes produced a similar decrease in total DNA binding. Chromatography of benzo[alpha]pyrene metabolite-DNA nucleoside adducts showed a marked reduction in four peaks and the elimination of one peak with microsomes from jacobine-treated rats.

Channel catfish liver monooxygenases. Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases.

Multiple drugs and pesticides are used in the aquaculture of channel catfish in the Southeastern United States. However, little is known regarding the enzymatic metabolism of these chemicals in the fish. Western blots, utilizing polyclonal antibodies raised against five purified rainbow trout liver cytochrome P450 enzymes, revealed at least two protein bands that were approximately 50 kDa (CATL-1) and 53 kDa (CATL-2). Anti-trout LMC3 and LMC4 only hybridized with the 53 kDa protein, whereas anti-trout LMC1, LMC2, and LMC5 recognized both proteins. Cytochrome P450-catalyzed activities (testosterone and progesterone hydroxylases) associated with LMC1 and LMC5 were also found in catfish liver microsomes. These data suggest that at least two constitutive forms of cytochrome P450 are present in the liver of juvenile channel catfish. Western blots utilizing antibodies raised against rabbit-lung flavin-containing monooxygenases (FMO) showed hybridization with two proteins from rainbow trout liver microsomes, but no cross-reaction with microsomes from catfish liver. N,N,-Dimethylaniline N-oxidase and methimazole oxidase were observed in microsomes from trout, but were absent in catfish liver microsomes prepared in three different laboratories. Consequently, FMO do not appear to be present in liver microsomes from channel catfish or they are rapidly degraded during tissue homogenization.

Hexachlorobenzene-induced porphyria in Japanese quail. Effect of pretreatment with phenobarbital or beta-naphthoflavone.

In an effort to determine the role that metabolism by the cytochrome P-450 system plays in the development of hexachlorobenzene (HCB)-induced porphyria, Japanese quail were pretreated with either beta-naphthoflavone (BNF) or phenobarbital (PB) and then treated with HCB. PB or BNF pretreatment appeared to have no effect on the response of quail hepatic enzymes to HCB. There were no differences between the two groups in either the content of cytochrome P-450 or the activities of NADPH-cytochrome c reductase, glutathione transferase (microsomal or cytosolic), ethoxycoumarin-O-deethylase or ethoxyresorufin-O-deethylase following HCB treatment. These pretreatments did, however, markedly influence the development of porphyria in quail. BNF-treated birds had higher delta-aminolevulinic acid-synthetase (ALA-S) activities and developed porphyria much more rapidly than birds treated with HCB alone. Birds pretreated with PB did not exhibit porphyria even following 10 days of HCB. Although the ALA-S activities in this group were elevated slightly following HCB, they were about one-half of those seen in the BNF-pretreated HCB-treated group. These results may reflect a difference between the PB and BNF groups in the production of a porphyrogenic metabolite of HCB.

Sex differences in cytochrome P-450 isozyme composition and activity in kidney microsomes of mature rainbow trout.

Kidney microsomes from sexually mature male, as opposed to female, rainbow trout displayed an approximately 20-fold higher cytochrome P-450 specific content, NADPH-cytochrome c reductase activity, and rates of hydroxylation of lauric acid, testosterone, progesterone and aflatoxin B1. Little or no sex difference in metabolism was observed with benzo[a]pyrene or benzphetamine as substrates. A similar pattern was observed in hepatic microsomes from these fish, but the difference was much less striking (approximately 2-fold higher activity in males). Juvenile trout (both sexes) possessed activities intermediate between mature males and females. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of kidney and liver microsomes of juvenile and sexually mature male and female trout suggested that the striking sex difference in kidney could be due to the high amount of trout P-450 isozyme LM2 in sexually mature males. Immunoquantitation of LM2, performed by Western Blotting and immunostaining with rabbit anti-trout LM2-IgG, confirmed that mature male kidney contained much higher levels of P-450 LM2 than juvenile or female kidney, or even of liver microsomes of all three groups. The amount of P-450 LM2 in mature female kidney microsomes was barely detectable. The high amount of LM2 in male trout kidney is consistent with the high activity of these microsomes towards lauric acid and aflatoxin B1, which have been shown previously to be preferentially metabolized by trout P-450 LM2. It is suggested that rainbow trout may serve as an alternative to the rat as an animal model for the study of sex-dependent differences in cytochromes P-450.

Role of metabolism in monocrotaline-induced immunotoxicity in C57BL/6 mice.

Monocrotaline (MCT) is a pyrrolizidine alkaloid which has been shown to induce immunotoxicity in mice. We hypothesized that metabolic activation of MCT by mixed-function oxygenases (MFO) to dehydromonocrotaline (MCTP) is a prerequisite for its immunotoxicity, as has been shown for other toxic effects of MCT. To test this hypothesis, we compared the in vitro immunotoxic potency of MCT and MCTP to suppress the in vitro antibody response to SRBC and the blastogenic response to B and T cell mitogens. In addition, the effects of in vivo modulation of MFO activities on the immunotoxicity of MCT was examined using phenobarbital (PB) to increase and chloramphenicol (CP) to decrease MCTP production. Results showed that in vitro exposure of splenic lymphocytes to MCT or MCTP produced significant suppression of the antibody and blastogenic responses. MCTP was 200-400-fold more potent than MCT. No metabolism of MCT by splenic cells was detectable, suggesting that unmetabolized MCT is capable of inducing immunotoxicity. In vivo studies showed that, while treatment of mice with PB or CP produced significantly increased and decreased MCTP production by liver microsomes, neither PB or CP treatment significantly altered the immunotoxic potency of MCT. Thus, while the MCTP metabolite is directly immunotoxic in vitro and much more potent than MCT, a role for the MCTP metabolite in MCT immunotoxicity in vivo could not be demonstrated.

Differential induction of hepatic drug metabolizing enzymes in Japanese quail by 1,2,4-trichlorobenzene.

The effects of single oral administration of 1,2,4-trichlorobenzene (TCB), 200, 400, 800 or 1600 mg/kg, and of daily oral administration of TCB, 400 mg/kg, for 3 consecutive days, on components of the microsomal monooxygenase system, glutathione, and the activities of cytosolic glutathione S-transferase and microsomal epoxide hydrolase in Japanese quail liver were studied. Cytochromes P-450 and b5 contents of liver microsomes and the activities of 7-ethoxyresorufin deethylase (7-ERD) and glutathione S-transferase were significantly increased 1 day after administration of single doses of TCB. Liver GSH and 7-ethoxycoumarin deethylase (7-ECD) activity were unchanged. Microsomal epoxide hydrolase activity was significantly decreased at TCB doses above 400 mg/kg. Increases in cytochromes and activities of 7-ERD and glutathione S-transferase were also seen following the 3-day administration of TCB, 400 mg/kg. In addition, liver GSH and the activity of NADPH-cytochrome c reductase were significantly increased whereas 7-ECD was significantly decreased by the 3-day treatment. These findings indicate that in Japanese quail, TCB is an inducer of 7-ERD and glutathione S-transferase but not of 7-ECD and epoxide hydrolase.

Studies on the porphyrinogenic action of 1,2,4-trichlorobenzene in birds.

The porphyrinogenic action of 1,2,4-trichlorobenzene (TCB) was examined in 17-day-old embryos, day-old chicks, 18-day-old chickens and adult Japanese quail. The quail was found to be the most sensitive species towards TCB induced porphyria whereas the chick embryo was totally non-responsive. The liver porphyrins of Japanese quail were increased in a dose-dependent manner 1 day after TCB. Elevation in porphyrin levels in quail was associated with comparable increases in delta-aminolevulinic acid synthetase (ALA-S) activity 1 day after TCB treatment. In contrast, ferrochelatase activity was found to be unchanged 1 day after TCB. Multiple administration of TCB produced only a slight increase in liver porphyrin levels and ALA-S activity in quail. However, there was a marked induction in ferrochelatase activity suggesting increased porphyrin turnover. Liver glutathione and glutathione S-transferase activity were also significantly increased following repeated administration of TCB in quail, which could indicate an enhancement of detoxication of reactive metabolites of TCB. Thus, it is suggested that the inability of low multiple doses of TCB to cause porphyria in Japanese quail may be related to the low responsiveness of ALA-S but high inducibility of ferrochelatase liver GSH and glutathione S-transferase.

Oregon's Toxic Household Products Law.

In 1991, Oregon became the first state in the U.S. to require the addition of an aversive agent to ethylene glycol-containing antifreeze and methanol-containing windshield wiper fluid. This new law, entitled "Toxic Household Products (THP) Act," was designed to reduce pediatric and animal poisonings from accidental ingestion of these two potentially lethal consumer automotive products. While not the stated intention of the law, addition of aversive agents to consumer automotive products could also reduce adult poisonings associated with intentional (suicides or alcoholics ingesting methanol-containing windshield wiper fluid) or accidental exposures. This law went into effect April 30, 1995, following settlement of a lawsuit brought by the Chemical Manufacturing Specialties Association (CSMA), a trade group representing the five largest manufacturers of ethylene glycol-based antifreeze in the U.S. This paper discusses the major policy issues that arose following the passage of Oregon's THP Act. Major provisions of the law are provided along with a discussion of CSMA's opposition to the Act's implementation. A description of the eventual settlement that was reached with CSMA as well as the major components of Oregon Health Division's (OHD) enforcement program are also highlighted. Data are presented for 1987 through 1998 on the number of exposures and severity of effects for pediatric cases (children < 6 years old) following exposure to both of these potentially lethal automotive products. However, because of the low incidence of exposures each year, these data are insufficient to draw any conclusions on the impact of the THP Act.

The formation of 6,7-dihydro-7-hydroxy-1-hydroxy-methyl-5H-pyrrolizine, a metabolite of pyrrolizidine alkaloids.

In vitro metabolism of senecionine with rat hepatic microsomes was studied. The main pyrrolic metabolite, 6,7-dihydro-7-hydroxy-1-hydroxy-methyl-5H-pyrrolizine was isolated by preparative high performance liquid chromatography (PRP-1 column) and examined by mass spectrometry. Results of the incubation performed in the presence of H218O indicated that both hydroxyl groups of the metabolite came from the solvent. Thus, formation of the pyrrolic metabolite is accomplished by alkyl-oxygen fission of both bonds C7-O and C9-O of the dehydroalkaloid, an intermediate supposedly formed during metabolic transformation of pyrrolizidine alkaloids (PAs).