Functional Monooxygenase Research Articles

Solubilization and characterization of a senecionine N-oxygenase from Crotalaria scassellatii seedlings

Abstract Seeds of Crotalaria scassellatii (Fabaceae) store pyrrolizidine alkaloids as tertiary amines. During the beginning of seed germination the tertiary alkaloids are rapidly converted into the respective alkaloid N -oxides which are the ultimate forms of alkaloid transport, metabolism and storage in vegetative plant organs. The enzyme catalyzing the N -oxidation was isolated from 2-day old seedling, partially purified and characterized. It is a membrane-bound, but not microsomal enzyme sedimenting in the 39 000 g fraction. The particulate enzyme was solubilized in the presence of 0.4% CHAPS and 0.4 M NaCl. The solubilized enzyme was partially purified (228-fold) by means of 70% ammonium sulfate precipitation and monocrotaline affinity chromatography. Inhibitor experiments, temperature sensitivity and lack of a carboxy ferrocytochrome absorption maximum at 450 nm strongly indicate SNO to be a flavin dependent enzyme. It is a mixed function monooxygenase that specifically N -oxidizes a number of structurally related pyrrolizidine alkaloids including the alkaloids of Crotalaria . A great variety of related alkaloids and xenobiotics were tested as substrates, none was accepted. The apparent K m values of senecionine, monocrotaline and heliotrine representing the three major types of pyrrolizidine alkaloids, are 12.4, 40.1 and 370.9 μ M, respectively. Senecionine is the best substrate, consequently the enzyme was named senecionine N -oxygenase (SNO). The substrate specificity of SNO is almost identical with that of a soluble insect SNO recently characterized from the haemolymph of arctiid larvae [Lindigkeit, R., Biller, A., Buch, M., Schiebel, H.-M., Boppre, M. and Hartmann, T., European Journal of Biochemistry , 1997, 245 , 626].

Is there a beneficial effect of the calcium channel blocker diltiazem on cyclosporine A nephrotoxicity in rats?

To investigate whether or not there is a beneficial effect of diltiazem (D) on cyclosporine A (CsA) nephrotoxicity, renal function, CsA blood levels, and effects of CsA on biotransformation in the liver and on lipid peroxidation were characterized in rats. A single administration of D (60 mg/kg b.wt.) reduced urinary volume (UV), GFR and excretion of Na+ and K+, whereas a single dose of CsA (60 mg/kg b.wt.) alone had no respective effects. P-aminohippurate excretion was almost equal in all groups. Lower doses of D (and CsA) were without effects. After repeated CsA treatment a retardation in body weight gain was seen, with little effect of a co-administration with D hereon. In all tests, thymus mass was reduced by CsA, the weight of spleen, liver, adrenal glands, and kidney were not generally affected by any of the treatments. Furthermore, after repeated administration of CsA and/or D, urinary volume, GFR and Na+ excretion were reduced by CsA, too. Electrolyte concentrations in plasma showed no evident changes by any of the treatments for Na+ and Ca2+. After long time treatment, CsA and CsA + D quite similarly led to higher K+ but lower Mg2+ concentrations in plasma. Only with 7 days highest dosage treatment PAH excretion was reduced significantly by CsA and CsA + D treatment. Surprisingly, CsA levels measured in blood and in kidney tissue, showed lower values after co-administration with D compared to CsA treatment alone. This could be caused by higher activities of monooxygenase functions revealed after pretreatment with D alone. Reduced glutathione (GSH) contents in kidney were elevated in CsA and CsA + D treated groups. In general no significant differences were to be observed concerning lipid peroxidation and stimulated H2O2 formation. Altogether evident protective effects of diltiazem on CsA nephrotoxicity in rats could not be proven.

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Developmental changes of cytochrome P450 dependentmonooxygenase functions after transplantation of fetal liver tissue suspension into spleens of adult syngenic rats

Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher 344 inbred rats. Animals were sacrificed at 3 days, 1, 2, 4 weeks, and 2, 4 and 6 months after transplantation and cytochrome P450 (P450) dependent monooxygenase functions in spleen and liver 9000 g supernatants were assessed by measuring three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; mainly 1A), ethoxycoumarin O-deethylation (ECOD; predominantly 1A, 2A, 2B) and ethylmorphine N-demethylation (END; mainly 3A). Values of transplant recipients were compared to those of sham operated and age matched control rats. Spleen weights were significantly higher in transplanted rats, compared to controls or sham operated animals, but there was no influence of the transplants within the spleens on liver weights. With fetal livers at the 21st day of gestation, the day of transplantation, a weak EROD and ECOD, but no END activity was seen. Spleens of controls or sham operated animals displayed nearly no P450 mediated monooxygenase functions. In the explant containing spleens a significant and increasing EROD activity was found from 4 weeks after surgery on and an ECOD activity already 2 weeks after transplantation. END was only slightly enhanced at 6 months after surgery. The livers of all three groups of rats displayed normal EROD, ECOD and END activities. Transplantation of fetal liver tissue suspensions into the spleens did not influence the P450 dependent monooxygenase functions within the livers of the animals. From these results it can be concluded that intrasplenically transplanted liver cells originating from syngenic fetal liver tissue suspensions proliferate and differentiate within the host organs. They display P450 dependent monooxygenase functions with some developmental changes during the observed time period of 6 months.

Effects of horminone on liver mixed function mono-oxygenases and glutathione enzyme activities of Wistar Rat

The present study reports on the effects of horminone on serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, on hepatic cytochrome P450 (P450) and cytochrome b 5 (cyt b 5) contents and on the activities of NADPH-cytochrome P450 reductase (NR), mixed function mono-oxygenases (MFO), glutathione- S-transferase (GST) and glutathione reductase (GR) of Wistar male rat. Horminone is a diterpenoid quinone (7,12-dihydroxyabiet-8,12-diene-11,14-dione) present in several species of the Labiatae family and used as medicinal plants in folk medicine. In this study, horminone was administered by the intraperitoneal route (i.p.) at a concentration of 1 or 10 mg/kg to each group of six mice, using water as a vehicle. On the one hand, results showed that horminone increased serum ALT and AST levels and cyt b 5 content and induced the activities of ethylmorphine N-demethylase (EMD). On the other hand, horminone decreased P450 content and inhibited the activities of 7-ethoxyresorufin O-deethylase (ERD), 7-ethoxycoumarin O-deethylase (ECD), aniline 4-hydroxylase (AH) and NR. Based on these results, the possibility of toxic effects occurring after administration of plant extracts containing horminone must be considered.

The two Faces of Pyrrolizidine Alkaloids: the Role of the Tertiary Amine and its N‐Oxide in Chemical Defense of Insects with Acquired Plant Alkaloids

Larvae of Creatonotos transiens (Lepidoptera, Arctiidae) and Zonocerus variegatus (Orthoptera, Pyrgomorphidae) ingest 14C-labeled senecionine and its N-oxide with the same efficiency but sequester the two tracers exclusively as N-oxide. Larvae of the non-sequestering Spodoptera littoralis eliminate efficiently the ingested alkaloids. During feeding on the two alkaloidal forms transient levels of senecionine (but not of the N-oxide) are built up in the haemolymph of S. littoralis larvae. Based on these results, senecionine [18O]N-oxide was fed to C. transiens larvae and Z. variegatus adults. The senecionine N-oxide recovered from the haemolymph of the two insects shows an almost complete loss of 18O label, indicating reduction of the orally fed N-oxide in the guts, uptake of the tertiary alkaloid and its re-N-oxidation in the haemolymph. The enzyme responsible for N-oxidation is a soluble mixed function monooxygenase. It was isolated from the haemolymph of the sequestering arctiid Tyria jacobaeae and purified to electrophoretic homogeneity. The enzyme is a flavoprotein with a native Mr of 200000 and a subunit Mr of 51000. It shows a pH optimum at 7.0, has its maximal activity at a temperature of 40-45 degrees C and an isoelectric point at pH 4.9. The reaction is strictly NADPH-dependent (Km 1.3 microM). From 20 pyrrolizidine alkaloids so far tested as substrates, the enyzme N-oxidizes only alkaloids with structural elements which are essential for hepatotoxic and genotoxic pyrrolizidine alkaloids (i.e. 1,2-double bond, esterification of the allylic hydroxyl group, presence of a second free or esterified hydroxyl group at carbon 7). A great variety of related alkaloids and xenobiotics were tested as substrate, none was accepted. The Km values of senecionine, monocrotaline and heliotrine, representing the three main types of pyrrolizidine alkaloids, are 1.3 microM, 12.5 microM and 290 microM, respectively. The novel enzyme was named senecionine N-oxygenase (SNO). The enzyme was partially purified from two other arctiids. The three SNOs show the same general substrate specificity but differ in their affinities towards the main structural types of pyrrolizidine alkaloids. The enzymes from the two generalists (Creatonotos transiens and Arctia caja) display a broader substrate affinity than the enzyme from the specialist (Tyria jacobaeae). The two molecular forms of pyrrolizidine alkaloids, the lipophilic protoxic tertiary amine and its hydrophilic nontoxic N-oxide are discussed in respect to their bioactivation and detoxification in mammals and their role as defensive chemicals in specialized insects. Pyrrolizidine-alkaloid-sequestering insects store the alkaloids as nontoxic N-oxides which are reduced in the guts of any potential insectivore. The lipophilic tertiary alkaloid is absorbed passively and then bioactivated by cytochrome P-450 oxidase.

Coexpression of a human P450 (CYP3A4) and P450 reductase generates a highly functional monooxygenase system in Escherichia coli

The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6β-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min −1, respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.

Coexpression of Mammalian Cytochrome P450 and Reductase inEscherichia coli

cDNAs for human cytochrome P450 2E1 and rat NADPH–cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins inEscherichia coliwas monitored by immunoblotting, spectroscopy, and catalytic assays. The cDNAs were separated on the coexpression plasmid by 22 nucleotides, with the P450 cDNA preceding the reductase cDNA. P450 content in solubilized cell membranes, whether expressed alone or coexpressed with P450 reductase, was approximately 0.11 nmol/mg of protein, and approximately 0.8 nmol could be obtained per liter of culture. Reductase content was five- to sixfold greater than P450 content when coexpressed, but severalfold less than that obtained when expressed without the upstream P450 cDNA, indicating differences in both stability and translatability between the two proteins. Solubilized membranes from cells expressing both proteins catalyzed aniline hydroxylation,p-nitrophenol hydroxylation, andN-nitrosodimethylamine demethylation at rates equivalent to those obtained by combining P450 and reductase preparations; addition of purified reductase to these membranes did not augment the activity. However, in contrast to results obtained with P450 2E1 expressed in other heterologous systems, addition of rabbit liver cytochromeb5to preparations catalyzingp-nitrophenol orN-nitrosodimethylamine oxidation did not increase turnover, and, although activity could be shown with unsolubilized membranes, oxidation of these substratesin vivocould not be demonstrated. Nonetheless, the ability to coexpress P450 and reductase inE. coliso as to generate a functional monooxygenase systemin vitroenhances the utility of this organism for the expression and characterization of cloned P450 isoforms.

Age related changes in NADPH cytochrome c reductase activity in mouse skin and liver microsomes.

In this study, the effect of ageing on the activity of cutaneous and hepatic NADPH cytochrome c reductase, a component of the mixed function mono-oxygenase system, was studied in young adult and senescent male C57B1/6J mice. Horse heart cytochrome c was used as substrate and its reduction by skin and liver microsomes was measured spectrophotometrically. Statistical analysis of our results show a significant decrease in cutaneous enzyme activity with age. This, however, was not the case in liver where no significant difference was seen, consistent with previous studies on mixed function mono-oxygenase activity in mouse liver. This suggests that the ageing process may have a tissue specific effect on the monooxygenase system in the C57B1/6J strain of mouse. However, further work in other rodent species and humans is required to fully characterize the effect of ageing on the drug metabolizing capacity of skin as compared to liver.

Correlation between cytochrome p450 2b1 induction by barbiturates and toxicity in the chick embryoin ovo

Using the highly sensitive probe‐substrate pentoxyresorufin towards cytochrome P450 2B1 isozymes, the relationship between the induction of this set of hemoproteins by barbiturates and toxicity was studied. Each chemical, allobarbital (AB), amobarbital (AM), barbital (BA), butobarbital (BB), buthetal (BU), cyclobarbital (CB), heptobarbital (HE), hexobarbital (HX), mephobarbital (ME), pentobarbital (PA), pentotal (PT), phenobarbital (PB), secobarbital (SE) and vinbarbital (VB) was injected in the chick embryo at the 16–18 day of embryonic incubation in equivalent doses (50, 25, 12.5, 6.25 and 3.12% of the respective LD50) and the mixed function monooxygenase activity examined in purified hepatic microsomes. For each barbiturate, linear regression was performed for both the inductive and toxic slope related to P450 2B1‐like activity in order to evaluate the IC100 in μmol/egg [the dose producing a 100 % increase of pentoxyresorufin dealkylation (PROD) during the induction phase] and the TC100, (the dose prod...

ALTERATION IN THE CAPACITIES OF CARCINOGEN METABOLIZING SYSTEM OF MOUSE-LIVERS DURING PRETREATMENT WITH VARIOUS ANTINEOPLASTIC AGENTS

The effect of treatment with antineoplastic drugs on the modification of the carcinog en-metabolizing capacity was studied in mice liver microsomes at different durations as a single and as a repeated dose treatment. It is generally demonstrated that there is a commonality of influence for each specific antineoplastic group on the expression of the mixed function mono-oxygenases. The antineoplastic alkylating agents (chlorambucil, melphalan and busulfan) significantly increased the activity of carcinogen metabolizing enzymes in contrary to the effects observed for the antibiotic actinomycin-D. The antimetabolite agents (5-flourouracil and methotrexate), on the other hand, although decreased these activities when administered as a single dose, a pronounced increase was observed at the repeated dose treatment. Significant increases in the activity of N-nitrosodimethylamine demethylases I and II and aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase were observed with chlorambucil when administered as a single or repeated doses. Similar effects was observed when the animals were treated with repeated doses of melphalan, busulfan and 5-flourouracil. Contrary to these effects, inhibition in the enzyme activity was exhibited when actinomycin-D was administered for either single or repeated dose treatment. The hepatic content of cytochrome P450 was significantly increased with all the administered drugs, except busulfan, when applied repeatedly. The implication of such alterations in the capacities of carcinogen metabolizing enzymes for antitumour-induced toxicity and carcinogenicity are discussed.

Method for estimation of benzo[ a]pyrene DNA adducts

Polycyclic aromatic hydrocarbons, e.g., benzo[ a]pyrene (B(a)P) are known carcinogens/mutagens. These compounds may be metabolized by the P450 mixed function monooxygenase to more nucleophilic compounds which may form adducts to the cellular macromolecules, e.g., DNA, RNA, and proteins. We have used synchronous fluorescence scanning for the assay of DNA adduct formation. In our earlier work with in vitro exposed human lymphocytes we estimated the adduct formation (femtomoles B(a)P per microgram DNA) to be higher than that estimated by other workers. We suggested that this difference may be related to the DNA isolation method used. In order to elucidate these differences we compared DNA adduct formation in human lymphocytes where DNA was isolated by the two different methods, i.e., using phenol extraction or the Gene Clean method. The data demonstrate that the phenol extraction procedure gives a yield of adducts per microgram DNA lower than that obtained by the Gene Clean method. The principle of the Gene Clean method for DNA isolation is protein denaturation by means of NaI followed by catching of DNA by absorption on silica particles. In contrast, the phenol extraction method is based upon phenol-mediated denaturation of proteins in the cell lysate leaving the hydrophilic nucleotides in the aqueous phase. However, during adduct formation more lipophilic adducts derived from DNA may redistribute between the aqueous phase and the phenol phase. In support of this theory we found higher adduct concentration per microgram DNA by the Gene Clean method 40 to 60 times than that found by the phenol method. Furthermore, assaying the distribution of adducts in both phases separated by the phenol method revealed an adduct concentration per microgram DNA in the phenol phase 34 times higher than that in the aqueous phase. Finally, it was shown by means of alkaline elution that the Gene Clean-isolated DNA contained single-stranded (adducted) DNA

Cytochrome P450IA2 in vivo induction: A potential biomarker of polyhalogenated biphenyls and their related chemical's effects on the human

The objectives of our studies have been to test the hypothesis that polyhalogenated biphenyls and related chemicals (PHB) can induce cytochrome P450 dependent mixed function monooxygenase family I enzymes (P450I) in the human, and the degree that a PHB induces P450I activity in the human correlates to the toxicity of the chemical. The P450I family enzyme, P450IA2, activity was monitored in the human by the caffeine breath test. The study subjects were exposed to high amounts of polybrominated biphenyls (PBB), polychlorinated biphenyls with polychlorinated dibenzofurans (PCB/DFs), or mirex. All the PHBs induced the P450IA2 activity of the exposed subjects as compared to the nonexposed control group. The capacity of each PHB to induce P450IA2 activity correlated with the known toxicity of each exposure ( PCB DF PBB >mirex ) in the human. P450IA2 activity as measured by the caffeine breath test may by a most useful biomarker in monitoring and understanding the effects of PHBs on the human population.

On the hepatotoxicity of 1,1,2,2-tetrachloroethane

Intoxication of male and female mice with a single dose (300 or 600 mg/kg) of 1,1,2,2-tetrachloroethane (TTCE) resulted in significant decreases in cytochrome P-450 (to 58–73% of the control) and NADPH-cytochrome ( P-450) c-reductase (to 29–35% of the control) in hepatic microsomes. This was accompanied by an alteration of mixed function monooxygenases stemming from the marked reduction (to 20–64% of the control) of several oxidative activities to selected substrates towards different P-450 isozymes (classes IA1, IA2, IIB1, IIE1 and IIIA). As phase II markers, epoxide hydrolase (≈35% loss), UDP-glucuronosyl transferase (≈42% loss) and to a lesser extent glutathione S-transferase (≈17% loss) were all affected. Also, the activity of δ-aminolevulinic (ALA) synthetase was decreased (≈57% of the control). On the contrary, heme oxygenase activity was increased (up to 35%) at the maximal dose tested. The decrease of P-450-function may be explained in terms of an alteration in the rate of heme biosynthesis and degradation, provoking a loss of heme content (≈33%) as well as of the direct inactivation of both P-450 and reductase. Because of increasing evidence on the involvement of free radical intermediates in the case of toxicity of haloalkanes, electron spin resonance spectroscopy (ESR) spin-trapping in vivo techniques were used to characterize the possible free radical species involved in the observed liver damage. The results obtained with the spin-trap N-benzylidene-2-methylpropylamine N-oxide (phenyl t-butylnitrone, PBN) provide evidence for the formation and trapping of the CHCl 2CHCl free radicals. The detection of conjugated diene signals by means of second-derivative spectrophotometry, have enabled us to show that in vivo lipid peroxidation may be one of the main mechanisms responsible for TTCE hepatotoxicity.

An emerging view of vitamin D

Vitamin D3 (D2 is 22-ene,24-methyl D3) is a prehormone which is hydroxylated by mixed function mono-oxygenase NADPH-cytochrome P-450 ferredoxin/ferredoxin reductase systems in liver parenchyma and renal proximal tubular cells to 25-hydroxy, then 1,25-dihydroxyvitamin D, the active hormone. 1,25-dihydroxyvitamin D binds to a mainly intranuclear receptor in target cells [classically, bone, kidney and gut; now shown to be wider including parathyroid cells, endocrine cells generally and many cells of ectodermal (brain, skin) and mesodermal (blood forming cells, lymphnode cells) origin as well as tumour cells (breast, lymphoma, leukaemia)] and activates transcription for products such as calcium binding proteins, its own receptor protein, 24-hydroxylase and non-specific esterase which are active in calcium homeostasis and cell differentiation. Advanced methods for measuring components of the vitamin D endocrine system have been developed and involve column extractions, liquid chromatographic purifications (also HPLC) and protein and receptor binding assays as well as mass spectrometry. These have facilitated elucidation of vitamin D physiology (also in pregnancy and lactation) and of metabolic defects in classical, vitamin D resistant and renal rickets and osteomalacia, in sarcoidosis and in the possible involvement of the vitamin in cell differentiation, e.g. in myeloid leukaemia, and breast cancer.

Correlations between hepatic monooxygenase system and glycogen storage

In experiments performed on female Wistar rats we have stated that simultaneously with glycogen mobilisation occurring immediately after epinephrine injection the amount of cytochrome P-450 (cP-450) was reduced. A characteristic cP-450 dependent function hexobarbital biotransformation was inhibited. No changes in hepatic protein and water content and serum bilirubin were measured. Glycogen and cP-450 loss and functional impairment depend on the administered epinephrine dose and time of treatment. We have proven that reduced activity of mixed function monooxygenase is related only to diminished glycogen content of the liver. Restitution of glycogen stores as well as amount and function of cP-450 were parallel.

Differences in H-2 recombinant mice in the beta-naphthoflavone inducibility of the mixed function monooxygenase, P1-450.

The influence of the major histocompatibility complex (H-2 in mouse) on induction of cytochrome P-450-dependent monooxygenase (P1-450) by the prototype polyaromatic hydrocarbon (PAH), beta-naphthoflavone, was investigated in C57BL/10 Sn (B10) recombinant congenic mice. The cytosolic Ah-receptor level, as measured by specific binding with [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin, was significantly lower in B10.A and B10.A(5R) than in either B10, B10.BR, or B10.A(2R), suggesting that the D region of H-2 influences Ah-receptor levels. The responsiveness to beta-naphthoflavone, as determined by increased catalytic activity toward benzo(a)pyrene and 7-ethoxycoumarin, was considerably lower in B10, B10.A, and B10.A(5R) than in B10.BR and somewhat lower than in B10.A(2R) or B10.A(4R) mice. The lower PAH responsiveness in B10.A and B10.A(5R) correlated with their lower Ah-receptor levels while that in B10 appeared to reflect a K-A region influence on PAH responsiveness that was not due to changed Ah-receptor levels. Thus, we conclude that more than one H-2 locus may influence PAH responsiveness, and by different mechanisms.

Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage.

AbstractFree radicals are chemical species characterized by an odd number of orbital electrons or by pairs of electrons of similar directional spin isolated singly in separate orbitals. Consequently most of these agents are highly reactive and usually exhibit an extremely short half‐life, although due to steric and resonance effects some exceptions occur. Some radicals and their precursors, such as the diradical O2 which exists in the triplet state, represent a critical and essential element of normal metabolism of aerobic organisms where, under normal circumstances, controlled reduction of reactive oxygen species occurs via the cytochrome oxidase or cytochrome P‐450 mixed function monooxygenase systems. In addition to reactive oxygen species, organisms may be subjected to a wide‐range of other free radicals or their precursors, including those of both exogenous and endogenous origin. Elaborate defense mechanisms have evolved to avoid cellular damage from these highly reactive species. Enzymes, such as the superoxide dismutase, the glutathione peroxidase/reductase system, and catalase; interactions with conjugated diene systems such as those found in melanins, carotenoids, and tocopherols; and direct reduction by sulphydryl compounds, phenols, and purines represent but a few of these natural defense systems.Despite a strong rationale for considering free radicals as pathologic agents, progress in implicating these agents, or their reactions, in pathologic processes has been arduous. The fore‐most hurdle to providing definitive evidence for free radical involvement rests with the highly transient nature of these species, hardly reaching measurable levels in vivo and thereby making rigorous testing of the hypothesis extremely difficult. Indeed, free radical damage has been studied, for the most part, by indirect means–usually by measurement of known free radical reaction intermediates and products from which free radical involvement is implied. Nevertheless, free radical formation has been shown to occur in UV‐irradiated skin and a considerable body of circumstantial evidence has been amassed that strongly infers that these agents, or reactions initiated by them, are responsible for at least some of the deleterious effects of UV upon skin.

Acute biological effects of commercial cresyl diphenyl phosphate in rats

A commercial cresyl diphenyl phosphate preparation was analyzed to contain approximately 35% of triphenyl phosphate, 45% of cresyl diphenyl phosphates, 18% of dicresyl phenyl phosphates and 2% of tricresyl phosphates. The product was almost free of the o-cresyl isomers as revealed by the analysis of its alkaline hydrolysis products. A single intraperitoneal injection (150 or 3 mg/kg) caused an induction of microsomal cytochrome P-450 in the liver of Wistar rats with a concomitant increase in the activities of mixed function monooxygenases and proliferation of smooth endoplasmic reticulum 24 h after the treatment. These effects were not detected in the kidneys. The morphological changes in hepatocytes included the enlargement of nuclei and mitochondria with increased cristae. The hepatic morphology returned to normal 2 weeks after the treatment. The activity of pseudocholine esterase in blood was inhibited 4 h and 24 h after the injection but the effect levelled off. The concentration of the organophosphates in blood and liver decreased rapidly with only traces detected in blood after 24 h. No effects on the activities of cerebral and muscle acetylcholine esterase were observed. The treatment (300 mg/kg) inhibited the brain — 2′,3′-cyclic nucleotide 3′-phosphohydrolase through the 2-week observation period associated with demyelination in peripheral nerves.

A monoclonal antibody against cytochrome P-450 enhances mutagen activation of N-nitrosodimethylamine by mouse liver S9: studies on the mode of action.

The effect of the monoclonal antibody MAb 2-66-3, directed against the major rat liver phenobarbital (PB)-induced cytochrome P-450 (P-450), on the S9-mediated mutagenicity of N-nitrosodimethylamine (DMN) in Salmonella typhimurium strain TA1530 was studied using liver S9 from PB-treated mice. This MAb enhanced approximately 2-fold S9-mediated mutagenicity of DMN but inhibited both its N-demethylation and N-denitrosation by 50%. Thus MAb-mediated enhancement of DMN mutagenesis does not result from altered activation/inactivation pathways, both known to involve P-450 isozymes. DMSO, a hydroxyl radical (HO.) scavenger and desferrioxamine, an inhibitor of HO.-dependent reactions, quenched the MAb-mediated enhancement of DMN mutagenesis, implicating the HO.-dependent activation of DMN to mutagenic species. As a mechanism, we propose that the binding of this MAb to P-450 isozyme implicated in DMN metabolism decreases the functional coupling between the reductase and the P-450 complex, leading to an increased electron flow from the reductase towards molecular oxygen to form reduced oxygen species (HO.) at the expense of the monooxygenase functions.