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J F Sinclair

Publications and source records attributed to J F Sinclair.

At least 19 recordsLinked to original sources
PubMed1999

Mutant yields and mutational spectra of the heterocyclic amines MeIQ and PhIP at the S1 locus of human-hamster AL cells with activation by chick embryo liver (CELC) co-cultures.

Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.

Animals↗
PubMed1998

Mechanism of the synergistic induction of CYP2H by isopentanol plus ethanol: comparison to glutethimide and relation to induction of 5-aminolevulinate synthase.

We had previously found that combined treatment with isopentanol and ethanol synergistically induced CYP2H protein and activity in cultured chick nepatoytes. Here we investigated the mechanism of induction of CYP2H by the alcohols and whether they caused a coordinate induction of 5-aminolevulinate synthase (ALAS) mRNA. Treatment with isopentanol alone or in combination with ethanol resulted in coordinate increases in CYP2H1 and ALAS mRNAs. With isopentanol alone, the amounts of CYP2H1 and ALAS mRNAs at 4 to 6 h were similar to those observed after treatment with the alcohol combination, but declined by 11 h. Readdition of isopentanol at 11 h again increased the expression of both mRNAs, indicating that the decreases at 11 h were due to limiting amounts of inducer. Similar results were observed in cells exposed to low concentrations of glutethimide. In the combined alcohol treatment, increases in CYP2H1 and ALAS mRNAs were sustained from 4 h to 11 h after addition of the alcohols, but decreased to control levels by 24 h. Using pulse labeling to measure de novo synthesis of CYP2H1/2 protein, we found that the increases in CYP2H1/2 protein reflected the increases in CYP2H1 mRNA. The half-life of CYP2H1/2 protein, measured from pulse-chase experiments, was approximately twofold greater than the half-life of CYP2H1 mRNA. Our results indicate that the alcohols and glutethimide coordinately increase ALAS and CYP2H1 mRNA, and that increases in CYP2H1/2 protein arise from increases in its mRNA.

5-Aminolevulinate Synthetase↗
PubMed1998

Induction of cytochrome P4503A by taxol in primary cultures of human hepatocytes.

In primary cultures of human hepatocytes, paclitaxel (Taxol), at pharmacological concentrations, was demonstrated to induce immunoreactive cytochrome P4503A (CYP3A). The magnitude of the inductive response of the hepatocytes to Taxol varied in five separate cultures. In general, exposure to increasing concentrations of Taxol (0.2 to 10 microM) resulted in increases in immunoreactive CYP3A. In four of the cultures, treatment of hepatocytes with the lowest concentration of Taxol tested (0.2 microM) resulted in approximately two-fold increases in CYP3A. In the other culture, however, a six-fold increase in CYP3A was observed at 0.2 microM. Taxol was almost as effective as rifampicin in inducing CYP3A in two of the cultures, but less effective than rifampicin in two other cultures. CYP3A4 mRNA was increased by Taxol. Increases in CYP3A4 mRNA correlated with increases in the levels of immunoreactive CYP3A. These results demonstrate that Taxol is a potent inducer of CYP3A in human hepatocytes. The clinical significance of these findings is discussed.

Adolescent↗
PubMed1998

Uroporphyria produced in mice by iron and 5-aminolaevulinic acid does not occur in Cyp1a2(-/-) null mutant mice.

In the present study we have investigated the putative requirement for the cytochrome P-450 isoform CYP1A2 in murine uroporphyria, by comparing Cyp1a2(-/-) knockout mice with Cyp1a2(+/+) wild-type mice. Uroporphyria was produced by injecting animals with iron-dextran and giving the porphyrin precursor 5-aminolaevulinic acid in the drinking water. Some animals also received 3-methylcholanthrene (MC) to induce hepatic CYP1A2. In both protocols, uroporphyria was elicited by these treatments in the Cyp1a2(+/+) wild-type mice, but not in the null mutant mice. Uroporphyrinogen oxidation activity in hepatic microsomes from untreated Cyp1a2(+/+) mice was 2.5-fold higher than in Cyp1a2(-/-) mice. Treatment with MC increased hepatic CYP1A1 in both mouse lines and hepatic CYP1A2 only in the Cyp1a2(+/+) line, as determined by Western immunoblotting. MC increased hepatic ethoxy- and methoxy-resorufin O-dealkylase activities in both mouse lines, but increased uroporphyrinogen oxidation activity in the Cyp1a2(+/+) wild-type mice only. These results indicate the absolute requirement for hepatic CYP1A2 in causing experimental uroporphyria under the conditions used.

Aminolevulinic Acid↗
PubMed1998

Formation of zinc protoporphyrin in cultured hepatocytes: effects of ferrochelatase inhibition, iron chelation or lead.

The formation of zinc protoporphyrin in response to lead or iron depletion has previously been investigated in erythroid systems. Because of its possible metabolic role in non-erythroid tissue, we investigated the formation of zinc protoporphyrin in cultured hepatocytes. The effects of lead and inhibitors of ferrochelatase, the iron insertion step of heme synthesis, on the conversion of 5-aminolevulinic acid to zinc protoporphyrin, protoporphyrin and heme were compared in rat and chick embryo hepatocyte cultures. In rat cultures, zinc protoporphyrin was synthesized enzymatically by ferrochelatase, since N-methylmesoporphyrin, an inhibitor of ferrochelatase. caused 40% or greater decreases in both heme and zinc protoporphyrin accumulation and markedly stimulated protoporphyrin accumulation. In addition, chelation of ferrous iron with 2,2'-dipyridyl decreased heme accumulation by 50%, but increased ZPP accumulation by 200%. Zinc protoporphyrin formation in chick embryo hepatocytes required the addition of zinc as well as 5-aminolevulinic acid and apparently was non-enzymatic, since it was not inhibited by N-methylmesoporphyrin nor increased by iron chelation. In the presence of 5-aminolevulinic acid, lead had no effect on zinc protoporphyrin, protoporphyrin or heme accumulation in chick hepatocytes, but decreased all three in rat hepatocytes, with the decrease in protoporphyrin being far greater than that of zinc protoporphyrin or heme. These findings indicate that, in contrast to the effect of lead in erythroid tissue, it did not specifically increase zinc protoporphyrin accumulation or alter iron availability in cultured hepatocytes.

Aminolevulinic Acid↗
PubMed1998

CYP1A-catalyzed uroporphyrinogen oxidation in hepatic microsomes from non-mammalian vertebrates (chick and duck embryos, scup and alligator).

Uroporphyrin (URO) accumulation in the liver of animals treated with polyhalogenated aromatic hydrocarbons (PHAH) is associated with increased microsomal oxidation of uroporphyrinogen catalyzed by rodent CYP1A2 and by a similar form in chicken, CYP1A5. The planar biphenyl, 3,3',4,4'-tetrachlorobiphenyl (TCB) stimulates uroporphyrinogen oxidation (UROX) in chick hepatic microsomes, but inhibits UROX activity in hepatic microsomes from mice and rats pre-induced by CYP1A2. Here we investigated whether TCB would stimulate or inhibit UROX in other non-mammalian species. UROX was stimulated 1.5-3-fold by TCB and 2-4-fold by 3,3',4,4',5,5'-hexachlorobiphenyl in hepatic microsomes from duck, alligator and scup treated with inducers of CYP1A. Hexachlorobenzene stimulated chick UROX, but was ineffective with microsomes from the other species. The stimulation of UROX by TCB was also observed in chick hepatocyte cultures. Pretreatment with up to 5 nM TCB induced CYP1A, but did not result in accumulation of URO. However, URO did accumulate if additional (post-induction) TCB was added along with 5-aminolevulinic acid. In this post-inductional TCB treatment, cycloheximide was included to prevent further induction of CYP1A. In duck hepatocytes, pretreatment with 25 nM TCB resulted in URO accumulation from 5-aminolevulinic acid. Post-induction TCB was not required and caused no further increase in URO accumulation. The differences in PHAH stimulation of UROX among the non-mammalian species have implications in the evolutionary changes in CYP1A, as well as the mechanism of development of PHAH-stimulated uroporphyria in different species.

Animals↗
PubMed1998

Uroporphyrinogen oxidation catalyzed by human cytochromes P450.

Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin. Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2. Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX). UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice. There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2. With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2. Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX. In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5. Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX. Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study. From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source. These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue. The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2.

Animals↗
PubMed1997

Kinetic mechanism of luciferase subunit folding and assembly.

The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha i and beta i. The alpha i beta i species can interact to form an inactive heterodimeric intermediate, [alpha beta ]i, which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 A resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956-21968]. In the absence of alpha i, beta i can form a kinetically trapped homodimer, beta 2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]; the structure of beta 2 has recently been reported [Thoden. J. B., Holden, H. M., Fisher, A. J., Sinclair. J. F., Wesenberg, G., Baldwin, T.O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta i species, or some other form that precedes beta i on the refolding pathway, can also undergo a first-order conversion into a form (designated beta x) that cannot associate with alpha i to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence of final yield on concentration of refolding species [Ziegler, M.M., Goldberg, M.E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol. Chem. 268, 10760-10765]. In simulations of the refolding reaction, all processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped-flow circular dichroism studies. The first-order rate constant for the folding of the alpha subunit, estimated from the concentration-independent lag preceding the appearance of active enzyme, and the second-order rate constant for assembly of alpha i and beta i into the heterodimer, estimated from the concentration-dependent rate of appearance of active enzyme, were consistent with the rates of first- and second-order processes monitored by changes in fluorescence of an extrinsic probe [the product of modification with N-(4-anilino-1-naphthyl)maleimide] on the alpha subunit during refolding. The rate constant for the isomerization of [alpha beta]i to form the active heterodimer was estimated from the kinetic data of a secondary dilution experiment and from fluorescence measurements of protein diluted 20-fold from 2.1 M urea-containing buffer. The rate constants reported here for the kinetic mechanism of refolding permitted simulation of the time courses and yields for activity recovery during the refolding of luciferase from about 1 to 25 micrograms/mL which are in excellent agreement with our previously reported data.

Circular Dichroism↗
PubMed1997

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

Biopolymers↗
PubMed1997

Effect of Taxol on cytochrome P450 3A and acetaminophen toxicity in cultured rat hepatocytes: comparison to dexamethasone.

The purpose of this study was to determine if Taxol induced CYP3A in primary cultures of rat hepatocytes and, if so, whether induction of CYP3A would increase acetaminophen toxicity. Taxol caused a concentration-dependent increase in the amount of immunoreactive CYP3A and in the steady-state levels of CYP3A1/DEX but not CYP3A2 mRNA. Similar concentration-dependent increases in toxicity as measured by a decrease in protein synthesis were observed after exposure of cells to acetaminophen for 7 hr whether cells were pretreated with Taxol or dexamethasone. Increased release of lactate dehydrogenase occured after 24 hr exposure to acetaminophen, with no further decreases in protein synthesis than those observed at 7 hr. Increases in acetaminophen toxicity correlated with increased covalent binding of acetaminophen to cellular proteins. Triacetyloleandomycin, a selective inhibitor of CYP3A, completely protected the cells against acetaminophen toxicity in both Taxol- and dexamethasone-pretreated cells and prevented the increase in covalent binding of acetaminophen to cellular proteins. These results demonstrate that Taxol, like dexamethasone, induces CYP3A and that increases in this P450 are responsible for increased acetaminophen toxicity.

Acetaminophen↗
PubMed1997

Role of CYP3A in ethanol-mediated increases in acetaminophen hepatotoxicity.

CYP2E is considered the only form of cytochrome P450 responsible for ethanol-mediated increases in acetaminophen hepatotoxicity. However, in experimental systems used for investigating ethanol-mediated increases in acetaminophen hepatotoxicity, animals are withdrawn from ethanol for 16 to 24 hr before the administration of acetaminophen to ensure the clearance of ethanol from the circulation. In rats, CYP2E has been shown to decrease to control levels after this time period of withdrawal from ethanol. We have previously shown in cultured human and rat hepatocytes, and in intact rats, that ethanol induces CYP3A in addition to CYP2E. To determine if there might be a role for CYP3A in ethanol-mediated APAP hepatotoxicity in addition to the recognized role for CYP2E, we investigated the effect of triacetyloleandomycin (TAO) on acetaminophen hepatotoxicity in ethanol-pretreated rats, as well as the effect of 11 hr withdrawal from ethanol on hepatic levels of CYP3A and CYP2E. TAO was dissolved in saline instead of dimethylsulfoxide, the solvent most usually employed, since dimethylsulfoxide inhibits CYP2E. Rats were administered 6.3% ethanol as part of the Lieber-DeCarli diet for 7 days, followed by replacement of the liquid diet with water for 11 hr. This 11-hr withdrawal from ethanol resulted in a decrease in hepatic levels of ethanol-induced CYP2E; however, considerable induction was still evident. There was no significant decrease in CYP3A. TAO completely prevented the histologically observed liver damage from acetaminophen in ethanol-pretreated rats, but did not prevent the increase in serum levels of AST. In ethanol-pretreated rats, exposure to APAP in the absence of TAO was associated with a 75% decrease in CYP3A, compared to animals exposed to APAP in the presence of TAO. These results suggest that CYP3A may have been suicidally inactivated by acetaminophen in the absence of TAO. Our findings suggest that CYP3A has a major role in ethanol-mediated increases in acetaminophen hepatotoxicity.

Acetaminophen↗
PubMed1997

Multiple roles of polyhalogenated biphenyls in causing increases in cytochrome P450 and uroporphyrin accumulation in cultured hepatocytes.

Uroporphyrin (URO) accumulation occurs in chick embryo hepatocytes treated with a number of polyhalogenated aromatic hydrocarbons (PHAHs) that are known inducers of cytochrome P4501As (CYP1A). Previous dose response studies had shown that URO accumulation does not begin until CYP1A, as indicated by ethoxyresorufin O-deethylase (EROD) activity, is maximally induced. The reason why the concentrations of PHAHs required for URO accumulation were higher than those required to induce EROD had not been explained. PHAHs, such as 3,3',4,4'-tetrachlorobiphenyl (PCB77, IUPAC nomenclature, TCB) stimulate uroporphyrinogen (UROGEN) oxidation by microsomes from 3-methylcholanthrene (MC)-treated chick embryos. Here we used a new protocol to investigate whether the requirement for more TCB to stimulate in vitro microsomal UROGEN oxidation extended to TCB-induced URO accumulation in intact cultured hepatocytes. Cultures were treated with increasing concentrations of TCB or other PHAHs to induce CYP1As, then with cycloheximide (CX) to prevent further P450 synthesis. The CX treatment was shown to block any further increases in CYP1A as determined by immunoblots. 5-Aminolevulinic acid and a high concentration of TCB ("postinduction TCB") were then added to stimulate intracellular UROGEN oxidation. Using the protocol with postinduction TCB, the inducing concentrations of TCB which caused URO to begin to accumulate were now much lower than in the absence of postinduction TCB. Increases in CYP1A proteins, measured immunochemically, were detected at about the same inducing TCB concentrations that began to increase URO accumulation. The new protocol, with postinduction TCB, using URO accumulation as the end point, greatly increased the sensitivity of the culture system for detection of PHAHs with EC50s (nM) for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), TCB, 3,3',4,4',5,5'-hexachlorobiphenyl, MC, and hexachlorobenzene being about 0.003, 0.11, 0.75, 3.5, and 30, respectively. As little as 2-4 fmol TCDD per culture dish caused detectible increases in URO accumulation. We conclude that URO accumulation in chick hepatocyte cultures is limited not only by the induction of CYP1A, but also by the stimulation of intracellular UROGEN oxidation.

Animals↗
PubMed1997

Ascorbic acid deficiency in porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), the most common form of porphyria, is manifested as skin photosensitivity caused by excess hepatic production of uroporphyrin and heptacarboxylporphyrin. In experimental animal models, ascorbic acid modulates chemically induced uroporphyrin accumulation. The purpose of this study was to determine whether ascorbic acid is decreased in the plasma of patients with PCT. Plasma was obtained after an overnight fast from 21 PCT patients, 16 of whom were infected with hepatitis C virus (HCV), and from a separate group of 9 patients with HCV infection but not PCT. Thirteen PCT patients were studied when they had active disease and 8 after treatment-induced remission. Plasma ascorbic acid was low (<23 micromol/L) in 11 (85%) of the 13 untreated PCT patients and deficient (<11 micromol/L) in 8 (62%). Two patients with normal ascorbic acid levels (45 and 62 micrommol/L) had consumed multivitamins. In 2 patients with deficient ascorbic acid, plasma levels returned to normal after phlebotomy treatment. Of the 8 patients studied during remission, 4 had normal ascorbic acid values and 4 were deficient (5 to 8 micromol/L). Plasma ascorbic acid values were normal for all patients who had HCV but no PCT. These data suggest that plasma ascorbic acid concentrations are commonly low in PCT, but this decrease is unrelated to HCV infection. Ascorbic acid deficiency may be one of the factors that contributes to the pathogenesis of PCT.

Adult↗
PubMed1997

Identification of CYP1A5 as the CYP1A enzyme mainly responsible for uroporphyrinogen oxidation induced by AH receptor ligands in chicken liver and kidney.

Uroporphyrinogen is an intermediate of the heme biosynthetic pathway. The oxidation of uroporphyrinogen to uroporphyrin (UROX) has been demonstrated to be catalyzed by mammalian CYP1A2. This reaction has an important role in uroporphyria caused by halogenated aromatic compounds. Two CYP enzymes induced by Ah receptor ligands were purified recently from chick embryo liver. One, designated CYP1A5, was preferentially active in arachidonic acid epoxygenation and the other, designated CYP1A4, in 7-ethoxyresorufin deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH), reactions mainly catalyzed by CYP1A1 in rodents. The amino acid sequences of both CYP1A5 and CYP1A4 are more similar to CYP1A1 than to 1A2, and neither can be classified as an ortholog of mammalian CYP1A1 or 1A2. Here we report that reconstituted purified CYP1A5 was eight times more active than CYP1A4 in catalyzing UROX. The stimulation of UROX by 3,4,3',4'-tetrachlorobiphenyl that has been observed in microsomes was also observed with the reconstituted enzymes. Similar dose response relationships were found for induction of UROX and EROD in both chick embryo liver microsomes and in cultured chick hepatocytes, indicating coinduction of CYP1A5 and CYP1A4. UROX was induced by the Ah receptor ligand, 3-methylcholanthrene, in chicken kidney as well as liver. The findings reported here and other evidence that CYP1A4 and CYP1A5 tend to exhibit CYP1A1 and 1A2-like enzyme activites, respectively, indicate that the division of some enzyme activities among CYP1A enzymes applies to different vertebrate classes.

Animals↗
PubMed1996

Oxidation of porphyrinogens by horseradish peroxidase and formation of a green pyrrole pigment.

When humans or plants are exposed to certain chemicals which interfere with heme biosynthetic enzymes, porphyrinogen intermediates accumulate and are oxidized to cytotoxic porphyrins. Here we have investigated the role of peroxidases in porphyrinogen oxidation. Horseradish peroxidase (HRP) rapidly oxidizes uroporphyrinogen to uroporphyrin and this is inhibited by ascorbic acid. HRP also oxidizes deuteroporphyrinogen (a synthetic porphyrin similar to protoporphyrinogen), but the yield of porphyrin is lower than with uroporphyrinogen as substrate. This low yield is in part due to a rapid, HRP-dependent conversion of deuteroporphyrin (but not uroporphyrin) to a green compound with spectral characteristics of a chlorin with a large peak at 638 nm. This reaction requires addition of a sulfhydryl reductant such as glutathione and is inhibited by ascorbic acid. These findings suggest that cellular peroxidases and ascorbic acid levels may play a role in modifying the phototoxic tetrapyrroles which accumulate in plants and humans after certain environmental exposures.

Horseradish Peroxidase↗
PubMed1996

Carbon monoxide dehydrogenase from Clostridium thermoaceticum: quaternary structure, stoichiometry of its SDS-induced dissociation, and characterization of the faster-migrating form.

The molecular mass (M(r)) of the nickel- and iron-sulfur-containing enzyme CO dehydrogenase from Clostridium thermoaceticum was determined by sedimentation equilibrium ultracentrifugation to be 300,000 +/- 30,000 Da. Since the enzyme is known to contain equal numbers of two types of subunits (M(r) = 82,000 Da for alpha and 73,000 Da for beta), this indicates an alpha 2 beta 2 quaternary structure. The enzyme was previously thought to have an alpha 3 beta 3 structure because it migrates through calibrated size-exclusion chromatographic columns with an apparent M(r) of about 420,000 Da. The disproportionately fast migration rate suggests that the enzyme is nonspherical. SDS induces the dissociation of an alpha subunit, yielding a stable species called FM-CODH. FM-CODH had a molecular mass of 210,000 +/- 30,000 Da, indicating an alpha 1 beta 2 structure. It contained 2.1 +/- 0.3 Ni and 16 +/- 3 Fe per alpha 1 beta 2, exhibited S-->Fe charge-transfer transitions typical of Fe-S proteins, and afforded the gav = 1.82, 1.86, and 1.94 EPR signals. Quantitation of the 1.82 and (1.94 +/- 1.86) signals afforded 0.35 and 1.9 spin/alpha 1 beta 2, respectively. FM-CODH samples exhibited CO oxidation activity, but little CO/acetyl-CoA exchange activity. Some FM-CODH samples exhibited CO oxidation activities as high as native enzyme. These results, along with the quantified spin intensities of the EPR signals, indicate that FM-CODH contains the B- and C-clusters and suggest that these clusters are located in the beta subunit. The alpha subunit that dissociated during formation of FM-CODH is not required for CO oxidation activity. FM-CODH is either devoid of A-clusters, or if such clusters are present, they have lost their ability to exhibit substantial NiFeC signals and CO/acetyl-CoA exchange activity. Incubating FM-CODH and alpha yielded a species that migrated through polyacrylamide gels at the same rate as native enzyme, and had a molecular mass indicating an alpha 2 beta 2 structure. Thus, the SDS-induced dissociation of the enzyme appears to be reversible.

Aldehyde Oxidoreductases↗
PubMed1995

Acute hepatotoxicity of acetaminophen in rats treated with ethanol plus isopentanol.

Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment.

Acetaminophen↗