In zebrafish, the onset of acetylcholinesterase (AChE) expression was detected by RT-PCR at 4 hpf (hours post-fertilization). The aryl acylamidase (AAA) associated with AChE, a serotonin sensitive activity with unknown physiological function, was significantly higher than the esterase activity on zebrafish embryos homogenates at 4-12 h development (test-t = 3.523; d.f. = 4). Remarkably, the ratio of AAA/AChE activity decreased 210-fold from 4 to 144 h development, indicating a distinct embryonic role of AAA during early embryogenesis. The AAA activity was sensitive to eserine and serotonin, ensuring its association with AChE. This is the first report of AAA activity on fish, establishing zebrafish as a model to study AAA on development.
The activation and covalent binding of 14C-labelled 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD) in mouse lung and liver S-9 preparations were examined in vitro. These results showed an oxidative cytochrome P-450 mediated transformation of o,p'-DDD to metabolite(s) that bind covalently to proteins, phospholipids and to added naked DNA in both lung and liver. The apparent Km-values for the covalent binding of o,p'-DDD to protein were 0.25 microM and 3.30 microM in lung and liver, respectively. Addition of glutathione to the incubation medium decreased the binding of o,p'-DDD more efficiently in the liver than in the lung. Thus, the selective lung binding of o,p'-DDD previously observed in vivo seems to result from an in situ activation. The tissue selectivity in vivo is suggested to be due to the low apparent Km in the lung favouring bioactivation at low, ecotoxicologically relevant doses, as well as to a less pronounced protection by glutathione in the lung.
Changes in the liver resulting from the low level dietary administration of 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU),p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE to Japanese Quail have been monitored. DDMU was exceptional in causing substantial increases in relative liver wt. and hepatic glucose-6-phosphatase after feeding at 100 ppm for 28 days. The time course of liver enzyme induction by DDMU has also been studied in Japanese Quail after periods of dietary administration ranging from 1--28 days with particular reference to changes in hepatic cytochrome P-450 and relative liver wt. Structural changes in the liver have been followed by reference to protein and lipid components. The hepatic response to DDMU appears to be biphasic. Initially there are substantial increases in hepatic cytochrome P-450 and relative liver wt., but the latter is largely due to accumulation of triglycerides. After approximately 20 days the level of hepatic cytochrome P-450 remain at a high 'plateau' level. This secondary phase of liver induction probably involves cell proliferation. It is concluded that DDMU causes major changes in the avian liver and either directly or through a metabolite causes pronounced microsomal enzyme induction.
The organochlorine insecticide 1,1'-(2,2,2-trichloroethylidene)bis(4-chlorobenzene) (DDT) did not induce or promote induction of morphological transformation in Syrian hamster embryo (SHE) cells, but it was a potent inhibitor of gap junctional intercellular communication (GJIC). The kinase inhibitor staurosporine did not affect DDT induced inhibition of GJIC, although it has been shown to decrease the inhibitory effect of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on GJIC. In addition, pretreatment with TPA made the cells refractory to further TPA induced inhibition of GJIC, while they remained sensitive to DDT. Thus, DDT and TPA inhibit GJIC through different mechanisms. Elevation of cellular cyclic adenosine monophosphate (cAMP) level by exposure to forskolin counteracted the inhibitory effect of DDT similar to that observed for TPA. Continuous exposure to DDT at concentrations near the effective concentration (50%) value (EC50 value) resulted in a slight recovery of GJIC following the initial inhibition. This recovery was not accompanied by the cells becoming refractory to further DDT induced inhibition of GJIC. The recovery of GJIC after removal of the DDT containing medium seemed to be related to a reduction in the amount of cell-associated DDT.
Dichloroacetaldehyde and 2,2-dichloro-1,1-dihydroxy-ethanephosphonic acid methyl ester which are formed solvolytically from desmethyltrichlorphone, an in vivo metabolite of the organophosphorus pesticide Trichlorphone, show in the dominant lethal test in mice at equimolar dosage (1.6 mmol/kg) a mutagenic activity comparable with that of Trichlorphon. Therefore it cannot be ruled out that the genetic effects of this pesticide may be due in part to the action of its degradation products.
1,1-Dichloroethylene (DCE) requires cytochrome P450-catalyzed bioactivation to electrophilic metabolites (1,1-dichloroethylene oxide, 2-chloroacetyl chloride and 2,2-dichloroacetaldehyde) to exert its cytotoxic effects. In this investigation, we examined the reactions of these metabolites with glutathione by spectroscopic and chromatographic techniques. In view of the extreme reactivity of 2-chloroacetyl chloride, primary reactions are likely to include alkylation of cytochrome P450, conjugation with GSH to give S-(2-chloroacetyl)-glutathione, or hydrolysis to give 2-chloroacetic acid. Our results showed conjugation of GSH with 1,1-dichloroethylene oxide, through formation of the mono- and di-glutathione adducts, 2-S-glutathionyl acetate and 2-(S-glutathionyl) acetyl glutathione, respectively. The observed equilibrium constant between the hydrate of 2,2-dichloroacetaldehyde and S-(2,2-dichloro-1-hydroxy)ethylglutathione was estimated from 1H-NMR experiments to be 14 +/- 2 M-1. Thus, 2,2-dichloroacetaldehyde is unlikely to make a significant contribution to GSH depletion as GSH concentrations above normal physiological levels would be necessary to form significant amounts of S-(2,2-dichloro-1-hydroxy)ethylglutathione. We also compared the formation of the glutathione conjugates in rat and mouse liver microsomes using 14C-DCE. The results demonstrated a species difference; the total metabolite production was 6-fold higher in microsomes from mice, compared with samples from rat. Production of DCE metabolites in hepatic microsomes from acetone-pretreated mice was 3-fold higher than those from untreated mice suggesting a role for P450 2E1 in DCE bioactivation. These results indicate that the epoxide is the major metabolite of DCE that is responsible for GSH depletion, suggesting that it may be involved in the hepatotoxicity evoked by DCE. Furthermore, this metabolite is formed to a greater extent in mouse than in rat liver microsomes and this difference may underlie the enhanced susceptibility found in the former species.
A series of new platinum(II) amine complexes containing 1,1- and 1,2-cyclobutanedicarboxylate ligands, cis-[PtA2(1,1-CBDCA)] (A = RNH2, where R = C2H5, n-C3H7, n-C4H9, n-C5H11, n-C6H13, c-C3H5, c-C5H9, c-C6H11; A2 = ethylenediamine, 1,3-diaminopropane), cis-[PtA2(1,2-CBDCA)] (A = NH3, RNH2 where R = CH3, C2H5, n-C3H7, n-C4H9, c-C3H5) and trans-[Pt(NH3)2(1,1-CBDCAH)2] (CBDCA, CBDCAH = dianion and monoanion of the dicarboxylic acid, respectively) have been synthesized by an improved route. These complexes are stable in aqueous solution and show good aqueous solubility. The [Pt(c-C3H5NH2)2(1,1-CBDCA)] can be isolated in white, grey and blue forms. The grey and blue forms exhibit ESR signals analogous to the so-called platinum blues. The existence of the blue form in aqueous solution is time and temperature dependent. Several of the complexes have been tested against leukaemia L1210 in male BDF mice and activity appears to decrease with the increase in length of the aliphatic chain (or increase in size of the alicyclic ring) of the primary amine. The Yoshida lymphoscarcoma screen, usually insensitive to platinum drugs, was found to respond well to [Pt(n-C4H9NH2)2(1,1-CBDCA)] in 5-day subcutaneously implanted tumours in female Wistar rats.
The metabolites of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) found in the urine of female Swiss mice are reported. The metabolites of DDT are DDD, 1-chloro-2,2-bis(p-chlorophenyl)ethene (DDMU), 1,1-dichloro-2-bis(p-chlorophenyl)ethene (DDE), 2,2-bis(p-chlorophenyl)acetic acid (DDA), 2-hydroxy-2,2-bis(p-chlorophenyl)acetic acid (alpha OH-DDA) and 2,2-bis(p-chlorophenyl)ethanol (DDOH), while DDD afforded DDMU, DDE, DDA, alpha OH-DDA and DDOH. The relative excreted levels of DDA and DDOH and the absence of 2,2-bis(p-chlorophenyl)acetaldehyde (DDCHO) are not consistent with the generally accepted pathway for DDA formation, which involves sequential metabolism of DDT and DDD via DDOH to afford DDA. The quantitative results are interpreted to mean that DDA is formed by hydroxylation at the chlorinated sp3-side chain carbon of DDD to give 2,2-bis(p-chlorophenyl)acetyl chloride (DDA-Cl), which in turn is hydrolyzed to DDA. The excretion of alpha OH-DDA from both DDT- and DDD-treated mice has never been previously observed. It is suggested that this metabolite arises from the initial epoxidation of DDMU, a metabolite of DDT and DDD, to yield 1,2-epoxy-1-chloro-2,2-bis(p-chlorophenyl)ethane (DDMU-epoxide). This chloroepoxide is then hydrolyzed and oxidized to produce the alpha OH-DDA.
Hepatic microsomal protein, cytochrome P-450, aniline hydroxylase, aromatic nitroreductase, phenyl benzoate esterase and total vitamin C, as well as tissue residue levels were measured following the feeding of low levels of 1,1-di(p-chlorophenyl)-2,2-dichloroethylene (DDE) or 1,1-di(p-chlorophenyl)-2,2,2-trichloroethane (DDT) to rats and Japanese quail. Changes in the factors measured were more readily related to the tissue residue levels than to the dietary feeding levels. It is suggested that DDE is a more potent inducer of microsomal protein, cytochrome P-450 and aniline hydroxylase in both species, than DDT fed at the same level, and the effects of DDT were best expressed in terms of the resulting DDE residue levels. Aromatic nitroreductase was not induced by feeding either DDT or DDE, and phenyl benzoate esterase levels showed a biphasic effect following DDE feeding. Vitamin C levels were raised more by DDT than DDE in both species. Residue levels were considerably higher in the quail than in the rat fed at the same rate. Basal levels of all the factors measured were higher in the rat and induction generally occurred to a greater extent and at a lower tissue residue level than in the quail.
Glutamate oxaloacetate transminase (GOT), glutamate dehydrogenase (GDH), sorbitol dehydrogenase (SDH), pseudo-cholinesterase (ChE) and various blood constituents were measured in the plasma of Japanese quail fed 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU) at low levels for periods ranging from 2 to 32 days. Previous work has shown that DDMU is a potent inducer of hepatic microsomal enzymes causing marked structural changes in the liver. A rapid increase in plasma GOT was observed within 4 days accompanied by an increase in relative liver weight. Plasma GDH and SDH increased to a maximum between 16 and 24 dyas which seems to be associated with hepatic cell proliferation. Plasma ChE showed a steady increase over the time course of DDMU administration. The level of plasma lipid was reduced after 4 days whereas the hepatic lipid content was substantially increased suggesting that the fatty liver condition may be caused by decreased release of triglyceride from the liver. Plasma glucose was reduced at 8 days but there was no evidence of a hyperglycaemic state. The changes noted after 2 days of DDMU diet were confirmed by measurements on birds 18 h after oral dosing the DDMU. The study demonstrates the value of plasma enzyme measurements for the early detection of toxic effects and indicates that DDMU administration leads to extrahepatic effects in addition to those previously described in the liver.
Hepatic microsomal protein, cytochrome P450, aniline hydroxylase and N-ethylmorphine demethylase as well as tissue residues were measured following the feeding of low levels of 1,1-di(p-chlorophenyl)-2,2-dichloroethylene (DDE) or 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU) to rats and Japanese quail. DDMU caused considerable elevation of the levels of most of the parameters measured in the quail even by comparison to the potent inducer, DDE, which gave greater tissue residues. In the rat where tissue residues of both DDE and DDMU were lower than those in quail, DDE caused greater changes in the measured enzyme levels than DDMU. Most of the changes caused by DDMU in the quail were larger than those observed following the ingestion of comparable levels of any other 1,1-di(p-chlorophenyl)-2,2,2-trichloroethane (DDT) metabolite in the rat or the quail. In the light of these and other published results it is suggested that the metabolic pathway for DDT in birds differs from that in mammals and probably gives rise through a pathway involving DDMU to a highly active liver inducer.
The strongly effective bactericidal compound 1,1'-hexamethylene-bis-[(5-p-chlorophenyl)-biguanide] (HCG), which is used as a disinfectant alterates the DNA of B. subtilis as shown in the rec assay, induced auxotrophic mutants in E. coli B and causes prophage induction in Micrococcus lysodeikticus 53-40 (N5). In vivo experiments with E. coli B have demonstrated that HCG extensively breaks down bacterial DNA and interacts with the synthesis of cellular DNA to the similar extent as found for N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The structural integrity of ribosomes and of ribosomal subunits remains intact in the presence of HCG.
A single dose of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (160 mg/kg i.p.) enhanced the monooxygenase step of drug biotransformation in rat liver. The O-demethylation of p-nitroanisole was especially increased, a peak in activity approximately 5-fold compared with controls being attained in 7 days. On the other hand, there was only a 2-fold increase in aryl hydrocarbon hydroxylase activity.DDT increased the cytochrome P-450 content of the liver, this increase coincided well with that in p-nitroanisole O-demethylation activity.The UDPglucuronosyltransferase activity of liver microsomes was not enhanced by DDT administration, unless the microsomes were pretreated to reveal latent activity prior to assay. After trypsin digestion of microsomes a maximum increase in activity of approximately 3-fold was observed as a result of DDT dosage. The canonic surfactant cetylpyridinium chloride was less active in revealing the latent UDP-glucuronosyltransferase activity, and two other membrane perturbants, the detergent digitonin and phospholipase A, were unable to show enhancement in UDPglucuronosyltransferase as a result of DDT dosage.
The susceptibility of polychlorinated ethanes to reductive metabolism was evaluated by measuring the amount of each compound consumed during anaerobic incubations with rat live microsomes; 1,1,1,2-tetrachloroethane, pentachloroethane and hexachloroethane were metabolized extensively, 1,1,1,2-tetrachloroethane and the trichloroethanes were metabolized very slowly and the dichloroethanes were not metabolized at a detectable rate. The electron affinity of the chloroethanes was determined by measuring electrochemical half-wave reduction potentials. Chloroethanes with an E1/2 of - 1.35 V or less negative were reduced readily in microsomes while those with an E1/2 equal to or more negative than -1.90 V were not good substrates for enzymatic reduction. Metabolites produced from 1,1,1,2-tetrachloroethane in vitro were 1,1-dichloroethylene (DCE) and 1,1,2-trichloroethane (TCEA) and the ratio DCE/TCEA was about 25:1. These conversions were NADPH-dependent and were inhibited by air, CO and metyrapone. In the presence of SKF 525-A, DCE formation was inhibited by 47%. Microsomes from untreated or beta-naphthoflavone-treated rats were 70-90% less active than microsomes from phenobarbital-treated rats. The Km was 0.50 mM and the Vmax was 66 nmol min-1 mg-1 protein for DCE formation. The results are consistent with the proposal that 1,1,1,2-tetrachloroethane is reduced by hepatic cytochrome(s) P-450 to a free radical intermediate which, for the most part, remains closely associated with the enzyme, is reduced further and undergoes beta-elimination of a chloride ion to form DCE. The occurrence of this reductive pathway in vivo was demonstrated by the quantitation of DCE and TCEA in blood from rats treated with 1,1,1,2-tetrachloroethane.
When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.
Structure-activity relationships have been studied for a series of doubly charged, fully co-ordinated, 1,10-phenanthroline transition metal chelates differing in the metal ion or in the number and nature of the substituent groups present on the 1,10-phenanthroline ligand by assaying their ability to inhibit the multiplication of influenza virus in chick allantoic cells in vitro. The most potent compounds are those derived from the highly labile metal ions Cd(II), Cu(II), Zn(II) and Mn(II) or highly lipophilic ligands. The rate of penetration of the active chelate species into the allantoic cell appears to be an important factor determining activity.
The actions of fully co-ordinated, inert nickel(II) chelates of 1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline have been compared on the guinea-pig isolated atrium with those of their constituent metal ion and ligands. In general, each test substance showed distinctive actions suggesting that the effects of the metal chelates are mediated by the intact chelate cation and not by liberated metal ion or ligand. Differences in the actions of the unsubstituted and the highly methylated compounds may result from the greater capacity of the methylated chelate and ligand to induce more profound and sustained conformational changes in the responsive atrial pre- and postsynaptic membranes thereby promoting more rapid and prolonged influx of Ca2+.
Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper-1,10-phenanthroline [(OP)(2)Cu(+)] complex was studied in situ. At 0.1 microM, MT inhibited the (OP)(2)Cu(+) induced DNA cleavage by about 50% (IC(50) approximately 0.1 microM). At 2.5 microM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu(+) (IC(50) of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP)(2)Cu(+) induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC(40) approximately 25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu(+)] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.
(1)Fully coordinated 1,10-phenanthroline and 2,2'-bipyridine chelates of Ru(II) are lethal in vitro to cultured and ascites P388 mouse lymphocytic leukaemic cells; 1,10-phenanthroline chelates are generally more potent than corresponding 2,2'-bipyridine compounds, and mixed-ligand (acetylacetonato) monovalent chelates of both series are more active than the corresponding identical-ligand divalent chelates. Lethal potency is greatest for Ru(II) chelates containing highly alkylated ligands. (2) Within two series of tetramethyl-1,10-phenanthroline chelates, the inert Ru(II) and Ni(II) members are less active against P388 cultured and ascites cells than the corresponding more labile chelates of Cu(II), Cd(II), Zn(II), Fe(II), and Co(II); for the ascites cells, the rank order of lethal potency of the chelates correlates reasonably well with their anticipated rank order of kinetic reactivity. (3) Repeated subculture of P388 cells in the presence of a mixed-ligand Ru(II) chelate has produced a cell line that shows a stable 10-fold resistance to the chelate; the resistant cell line is selectively cross-resistant to certain Ru(II) identical and mixed-ligand chelates. (4) The presence of a fluorescent Ru(II) chelate has been demonstrated at the surface and within the cytoplasm and nucleus of P388 ascites cells exposed to it either in vitro or in the mouse. (5) Ru(II) and Cu(II) chelates of tetramethyl-1,10-phenanthroline do not appear to be chemotherapeutically active against P388 ascites cells in the mouse.
The anti-cancer chemotherapeutic potential of 1,10-phenanthroline-5,6-dione (phendione), [Cu(phendione)(3)](ClO(4))(2).4H(2)O and [Ag(phendione)(2)]ClO(4) were determined using four human cells lines, i.e. two neoplastic (A-498 and Hep-G2) and two non-neoplastic (CHANG and HK-2). All of the phendione derivatives induced a concentration-dependant decrease in the viability of the four cell lines, with [Cu(phendione)(3)](ClO(4))(2).4H(2)O displaying greatest activity. In comparative studies, IC(50) values obtained with the two neoplastic cell lines showed a cytotoxic response which was between 3 and 35 times greater than that observed for the metal-based anti-cancer agent, cisplatin. Furthermore, metal-phendione complexes, rather than simple solvated metal ions, were responsible for the observed cytotoxicity. Despite the high level of potency associated with these compounds they did not display an apparent cyto-selective profile, as they reduced the viability of both neoplastic and non-neoplastic cells. However, selected mechanistic studies showed that phendione and its metal complexes inhibited DNA synthesis which did not appear to be mediated through intercalation. Ames testing highlighted that all three compounds and their phase I metabolites were non-mutagenic, unlike cisplatin. Taken together, these results suggest that phendione and its Cu(II) and Ag(I) complexes may be capable of acting as highly effective anti-cancer therapies, which with careful administration could provide very potent and effective alternatives to cisplatin.
The actions of two related series of fully co-ordinated, divalent 1,10-phenanthroline transition metal chelates have been investigated on the rat isolated diaphragm muscle-phrenic nerve preparation and, where possible, compared with those of their constituent metal ions and ligands. Each member of both series of chelates produced blockade of neuromuscular transmission, although mechanistically not of a uniform type, and several elicited varying degrees of muscle contracture. The kinetic reactivity of the metal chelate appeared to be an important factor determining the nature of the biological response and profound differences in response were observed between labile and inert chelates differing in some cases by only one electron in the 3d shell.
Naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are the major metabolites of naphthalene that are thought to be responsible for the cytotoxicity and genotoxicity of this chemical. We measured cysteinyl adducts of these metabolites in hemoglobin (Hb) and albumin (Alb) from F344 rats dosed with 100-800 mg naphthalene per kg body weight. The method employs cleavage and derivatization of these adducts by trifluoroacetic anhydride and methanesulfonic acid followed by gas chromatography-mass spectrometry in negative ion chemical ionization mode. Cysteinyl adducts of both proteins with NPO, and 1,2- and 1,4-NPQ (designated NPO-Hb and -Alb, 1,2-NPQ-Hb and -Alb, and 1,4-NPQ-Hb and -Alb, respectively) were produced in a dose-dependent manner. Of the two structural isomers resulting from NPO, levels of NPO1 adducts were greater than those of NPO2 adducts in both Hb and Alb, indicating that aromatic substitution is favored in vivo at positions 1 over 2. Of the quinone adducts, 1,2-NPQ-Hb and -Alb were produced in greater quantities than 1,4-NPQ-Hb and -Alb, indicating either that the formation of 1,2-NPQ from NPO is favored or that more than one pathway leads to the formation of 1,2-NPQ. The shapes of the dose-response curves were generally nonlinear at doses above 200 mg naphthalene per kg body weight. However, the nature of nonlinearity differed, showing evidence of supralinearity for NPO-Hb, NPQ-Hb and NPQ-Alb and of sublinearity for NPO-Alb. Low background levels of 1,2-NPQ-Hb and -Alb and 1,4-NPQ-Hb and -Alb were detected in control animals without known exposure to naphthalene. However, the corresponding NPO-Hb and -Alb adducts were not detected in control animals.
The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2-BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF-induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2-DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes.
3-Butene-1,2-diol (butenediol), a major metabolite of 1,3-butadiene (butadiene), can undergo either detoxification or biotransformation to potentially toxic metabolites, including 3,4-epoxy-1,2-butanediol and hydroxymethylvinyl ketone (HMVK). Butadiene exposure can occur concomitantly with hexanes, which share common biotransformation pathways with butadiene. To determine the potential influence of hexane co-exposure on butadiene toxicity, the present study examined the effect of n-hexane on butenediol disposition [as measured by urinary excretion of (N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine) (MI level)] and genotoxicity (as measured by the frequency of bone marrow micronucleated erythrocytes) and acute toxicity (as measured by body weight changes) in the rat. The results show that butenediol was not genotoxic to adult or immature rats but was acutely toxic to adult but not immature rats. The results also suggest that n-hexane co-exposure may attenuate the acute toxicity by butenediol in adult rats and that immature rats may be less sensitive than adults to the acute toxicity.
The ability of naphthalene 1,2-oxide to diffuse across intact cellular membranes, the subsequent biotransformation of this epoxide and its potential to produce losses in cellular viability have been examined in incubations of isolated hepatocytes. Addition of 1R,2S- or 1S,2R-naphthalene oxide enantiomers (15, 30 and 60 microM) to isolated hepatocytes resulted in a rapid depletion of intracellular glutathione. Depletion of glutathione was concentration dependent and maximal at 5-15 min. Addition of either of the enantiomeric oxides at 60 microM resulted in the loss of more than 20 nmol glutathione/10(6) cells (1 ml cells); thus more than a third of the added epoxide was available for conjugation with intracellular glutathione. The time course and concentration dependence of glutathione depletion corresponded to the rapid, concentration-dependent formation of naphthalene oxide glutathione conjugates. The levels of glutathione adduct were highest 1 min after addition of naphthalene oxide and declined to 25% of this level after 30 min. Loss of glutathione conjugates from incubations correlated with the formation of N-acetylcysteine adducts. In contrast, the levels of glutathione adducts added exogenously to hepatocytes were relatively stable over a 120-min incubation suggesting that although further metabolism of naphthalene oxide glutathione adducts formed intracellularly is possible, extracellular glutathione adducts cannot penetrate the hepatocellular membrane. Small amounts of radiolabel from [3H]naphthalene 1,2-oxide were bound covalently to macromolecules in hepatocytes; the rate of this binding slowed rapidly after the first minute of incubation. Severe blebbing of the surface of the hepatocytes was noted in cells incubated for 30 min with 480 microM naphthalene oxide. Many of the cells were vacuolated at 60 min and progressed to frank necrosis with pyknotic nuclei and inability to exclude trypan blue. Cells incubated with 1-naphthol responded in a qualitatively similar fashion to those cells incubated with epoxide; however, hepatocytes incubated with 1-naphthol progressed to frank cellular necrosis at a slower rate. In hepatocytes partially depleted of glutathione by pretreatment with buthionine sulfoximine, addition of 1S,2R-naphthalene oxide at a rate of 1 nmol/min/10(6) cells resulted in significant losses in cell viability. In contrast, no losses in cell viability were observed with the enantiomer, 1R,2S-naphthalene oxide. Both epoxides produced similar losses in cellular glutathione levels.(ABSTRACT TRUNCATED AT 400 WORDS)
Butadiene (BD) metabolism shows gender, species and concentration dependency, making the extrapolation of animal results to humans complex. BD is metabolized mainly by cytochrome P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2;3,4-diepoxybutane (DEB) and 1,2-epoxy-butanediol (EB-diol). For accurate risk assessment it is important to elucidate species differences in the internal formation of the individual epoxides in order to assign the relative risks associated with their different mutagenic potencies. Analysis of N-terminal globin adducts is a common approach for monitoring the internal formation of BD derived epoxides. Our long term strategy is to develop an LC-MS/MS method for simultaneous detection of all three BD hemoglobin adducts. This approach is modeled after the recently reported immunoaffinity LC-MS/MS method for the cyclic N,N-(2,3-dihydroxy-1,4-butadyil)-valine (pyr-Val, derived from DEB). We report herein the analysis of the EB-derived 2-hydroxyl-3-butenyl-valine peptide (HB-Val). The procedure utilizes trypsin hydrolysis of globin and immunoaffinity (IA) purification of alkylated heptapeptides. Quantitation is based on LC-MS/MS monitoring of the transition from the singly charged molecular ion of HB-Val (1-7) to the a(1) fragment. Human HB-Val (1-11) was synthesized and used for antibody production. As internal standard, the labeled rat-[(13)C(5)(15)N]-Val (1-11) was prepared through direct alkylation of the corresponding peptide with EB. Standards were characterized and quantified by LC-MS/MS and LC-UV. The method was validated with different amounts of human HB-Val standard. The recovery was >75% and coefficient of variation <25%. The LOQ was set to 100 fmol/injection. For a proof of principal experiment, globin samples from male and female rats exposed to 1000 ppm BD for 90 days were analyzed. The amounts of HB-Val present were 268.2+/-56 and 350+/-70 pmol/g (mean+/-S.D.) for males and females, respectively. No HB-Val was detected in controls. These data are much lower compared to previously reported values measured by GC-MS/MS. The difference may be due higher specificity of the LC-MS/MS method to the N-terminal peptide from the alpha-chain versus derivatization of both alpha- and beta-chain by Edman degradation, and possible instability of HB-Val adducts during long term storage (about 10 years) between the analyses. These differences will be resolved by examining recently collected samples, using the same internal standard for parallel analysis by GC-MS/MS and LC-MS/MS. Based on our experience with pyr-Val adduct assay we anticipate that this assay will be suitable for evaluation of HB-Val in multiple species.
It has been widely recognized that induction of Phase 2 enzymes is an effective and sufficient strategy for achieving protection against carcinogenesis. Nrf2 is the unifying master regulator of these enzymes and its activation in various tissues, including the urinary bladder, is associated with inhibition of carcinogenesis. 5,6-Dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) is a highly potent inducer of Phase 2 enzymes and an activator of Nrf2. In vivo, it is particularly effective in the bladder, showing significant effects in this tissue when dosed to rats at levels as low as 0.98 micromol/(kgday) (0.17 mg/(kg day)). The activities of key Phase 2 enzymes, including glutathione S-transferase, NAD(P)H:quinone:oxidoreductase 1 and glutamate cysteine synthetase, and levels of glutathione were elevated by CPDT in rat bladder in vivo and in cultured bladder cells in vitro. In the bladder, enzyme induction and Nrf2 activation appear to occur exclusively in the epithelium. This is highly significant, since almost all bladder cancers develop from the epithelium. Studies in cultured bladder cells using siRNA to knock down Nrf2 or in cells with total Nrf2 knockout showed that the ability of CPDT to induce Phase 2 enzymes depends completely on Nrf2. In conclusion, CPDT potently and preferentially induces Phase 2 enzymes in the bladder epithelium and Nrf2 is its key mediator.
Biotransformation of drugs and environmental chemicals to reactive intermediates is often studied with the use of radiolabeled compounds that are synthesized by expensive and technically difficult procedures. In general, glutathione (GSH) conjugation serves as a detoxification mechanism, and conjugation of reactive intermediates with GSH is often a surrogate marker of reactive species formation. However, several halogenated alkanes can be bioactivated by GSH to yield highly reactive GSH conjugates, some of which are DNA-reactive (e.g. conjugates of 1,2-dibromoethane). The purpose of this study was to metabolically radiolabel the in vivo GSH pool of Salmonella typhimurium with a [35S]-label and to examine the GSH-mediated bioactivation of a model haloalkane, 1,2-dibromoethane, by measuring the binding of [35S]-label to DNA. The strain of Salmonella used in this study had been transformed previously with the gene that codes for rat glutathione transferase theta 1-1 (GSTT1-1), an enzyme that can catalyze formation of genotoxic GSH conjugates. Bacteria were grown to mid-log phase and then incubated with [35S]-L-cysteine in minimal medium (thio-free) until stationary phase of growth was reached. At this stage, the specific activity of Salmonella GSH was estimated to be 7.1 mCi/mmol by derivatization and subsequent HPLC analysis, and GSTT1-1 enzyme activity was still demonstrable in Salmonella cytosol following growth in a minimal medium. The [35S]-labeled bacteria were then exposed to 1,2-dibromoethane (1 mM), and the Salmonella DNA was subsequently purified to quantify [35S]-binding to DNA. The amount of [35S]-label that was covalently bound to DNA in the GSTT1-1-expressing Salmonella strain (33.2 nmol/mg DNA) was sevenfold greater than that of the control strain that does not express GSTT1-1. Neutral thermal hydrolysis of the DNA yielded a single [35S]-labeled adduct with a similar t(R) as S-[2-(N(7)-guanyl)ethyl]GSH, following HPLC analysis of the hydrolysate. This adduct accounted for 95% of the total [35S]-label bound to DNA. Thus, this [35S]-radiolabeling protocol may prove useful for studying the DNA reactivity of GSH conjugates of other halogenated alkanes in a cellular context that maintains GSH at normal physiological levels. This is also, to our knowledge, the first demonstration of de novo incorporation of [35S]-L-cysteine into the bacterial GSH pool.
Polycyclic aromatic hydrocarbon (PAH) o-quinones are products of an NADP+ dependent oxidation of non-K-region trans-dihydrodiols catalyzed by dihydrodiol dehydrogenase (EC 188.8.131.52). Since these PAH o-quinones could be detoxified by non-enzymatic or enzymatic conjugation with cellular thiols, their reactivity with 2-mercaptoethanol, cysteine and glutathione (GSH) was examined by ion-pair reverse phase high pressure liquid chromatography (RP-HPLC). Second-order rate constants for the addition of these thiols to naphthalene-1,2-dione (NPQ) in water ranging from 4.9 x 10(3) - 1.1 x 10(4) min-1 M-1 and the reactions were complete within 10 min. When these reactions were conducted at near physiological pH (50 mM potassium phosphate buffer pH 7.0), the rate constants increased by 2-orders of magnitude. When benzo[a]pyrene-7,8-dione (BPQ) was substituted in these reactions the second-order rate constants decreased by 2-3 orders of magnitude and the reactions took several hours to reach completion. The decrease in reactivity can be explained by the presence of the bay region in BPQ. Methylation influenced the reactivity of PAH o-quinones with GSH and the following order of reactivity was observed: 7,12-dimethyl-benz[a]anthracene-3,4-dione (7,12-DMBAQ) > 12-methyl-BAQ, 7-methyl-BAQ and BAQ > BPQ. Of these quinones 7,12-dimethyl-BAQ was almost equi-reactive with NPQ. This suggests that methyl substitution in the bay and peri regions enhances reactivity with GSH. Using NPQ as a model for other PAH o-quinones, N-acetyl-L-cysteine, L-cysteine and GSH conjugates of NPQ were synthesized and characterized by [1H]- and [13C]NMR. Evidence for Michael type 1,4-addition products was obtained in which the resultant adduct could exist as either a catechol or o-quinone. By contrast, L-cysteine was able to form adducts via S- or N-attack and N-attack gave a purple p-iminoquinone. There was no evidence for the formation of bis-N-acetyl-L-cysteinyl-, bis-glutathionyl adducts or phenolic coupled products. The toxicity of thiol conjugates of NPQ remains to be explored.
1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds, into which they decompose on thermal or photochemical activation. In the presence of DNA, the decomposition of dioxetanes gives rise to DNA modifications, which have been studied by means of specific repair endonucleases. Cyclobutane pyrimidine dimers, which are generated by triplet-triplet energy transfer, were detected by a UV endonuclease; they made up between 2% and 30% of the total modifications recognized by a crude repair endonuclease preparation from Micrococcus luteus. For various 1,2-dioxetanes, the yield of pyrimidine dimers was proportional to their triplet excitation flux. DNA strand breaks, sites of base loss (AP sites; recognized by exonuclease III and endonuclease IV) and dihydropyrimidines (recognized by endonuclease III) were found to represent only a small fraction of the modifications. The majority of the modifications detected were recognized by formamidopyrimidine-DNA glycosylase (FPG protein) and represent 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) residues or other yet not defined base modifications which are recognized by this enzyme. The modifications were generated in similar relative yields by thermal and photo-induced decomposition of the 1,2-dioxetanes and therefore emanate under both conditions from the excited carbonyl compounds. The formation of the FPG protein-sensitive modifications was efficiently quenched by azide anions; the Stern-Volmer quenching of these modifications was 150-fold more effective than that of the pyrimidine dimers. The relative amounts of the two types of modifications were strongly dependent on the structure of the 1,2-dioxetanes and on the concentration of molecular oxygen. Singlet oxygen appears to be involved only to some extent in the generation of the FPG protein-sensitive base modifications as their yield was only moderately (approximately 2-fold) increased in D2O as solvent. A mechanism is suggested in which oxidized guanine is predominantly formed by a single-electron-transfer reaction of the triplet excited carbonyl product derived from the 1,2-dioxetane, followed by unknown secondary oxidations, which involve molecular oxygen and/or undecomposed 1,2-dioxetane.
Metabolic activation by cytochrome P-450 of glutamic acid pyrolysis products, 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole (Glu-P-1) and 2-amino-dipyrido(1,2,-a:3',2'-d)imidazole (Glu-P-2), to mutagenic metabolites was studied using Salmonella typhimurium TA98 as a tester strain. Cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH were essential requirements for the activation of these compounds. Of the four forms of cytochrome P-450 examined, polychlorinated biphenyls (PCB) P-448 and 3-methylcholanthrene (MC) P-448 purified from liver microsomes of rats treated with a PCB mixture and MC, respectively, showed high activity in the activation of both Glu-P-1 and Glu-P-2. The presence of three metabolites from Glu-P-1 or Glu-P-2 was demonstrated by high performance liquid chromatographic (HPLC) analysis. Among the metabolites of Glu-P-1, two metabolites were mutagenic without any further enzymatic activation. In accordance with the results of a mutation assay, PCB P-448 also exhibited higher activity to form the major mutagenic metabolite of Glu-P-1. The major active metabolite of Glu-P-1 was characterized as N-hydroxy-Glu-P-1 by chemical analysis using oxidizing and reducing reagents and by mass spectrometry.
In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.
In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.
The mutagenic activities in V79 Chinese hamster cells and the alkylating abilities towards nicotinamide of the two diastereisomeric cis and trans-3-bromo-1,2-epoxycyclohexanes were measured and compared with those of unsubstituted 1,2-epoxycyclohexane and bromocyclohexane. trans-3-Bromo-1,2-epoxycyclohexane exhibited a mutagenic activity 2.5 times higher than that of its cis diastereoisomer, but very similar to that of the parent unbrominated epoxide, whereas the electrophilic reactivities towards nicotinamide were very similar for the three epoxides tested. Bromocyclohexane showed the highest toxicity, but no alkylating ability. The presence of an epoxide hydrolase activity in the V79 Chinese hamster cells used in the mutagenesis tests has been demonstrated using safrole oxide as the substrate, cis-3-Bromo-1,2-epoxycyclohexane, but not its trans diastereoisomer, is hydrolyzed by the enzyme present in microsomal preparations from the V79 cells. The results indicate that for the cycloaliphatic compounds examined: (1) the introduction of a bromide substituent at the carbon adjacent to the oxirane ring does not cause an increase in mutagenicity, (2) the relative stereochemical configuration at the above carbon does affect the biological activity and (3) the significantly different mutagenicity of the two diastereoisomeric 3-bromo-1,2-epoxycyclohexanes is not attributable to a different electrophilic reactivity, but could be related to some specific interaction with detoxifying enzymes present in the V79 Chinese hamster cells used in the biological experiments.
The metabolism and genotoxicity of 1,2-dibromoethane (EDB) and its deuterium substituted analog ( d4EDB ) were studied in isolated rat hepatocytes. There was a marked isotope effect on the metabolism of EDB by hepatocytes. This was due to decreased microsomal oxidation of d4EDB . Cytosolic metabolism of EDB, as measured by bromide ion release, was unaffected by deuterium substitution. The genotoxicity of the two analogs was assessed by assaying for the presence of EDB induced single-strand breaks in DNA. As measured by the alkaline elution technique, both compounds caused DNA single-strand breaks when incubated at a concentration of 0.1 mM with hepatocytes. No difference in the degree of DNA damage could be demonstrated between hepatocytes incubated with EDB or d4EDB . These data suggest that the GSH transferase mediated metabolism of EDB is responsible for the genotoxic effects of EDB observed in hepatocytes.
Lung cancer continues to be the leading cause of cancer deaths throughout the world and conventional therapy remains largely unsuccessful. Although, chemoprevention is a plausible alternative approach to curb the lung cancer epidemic, clinically there are no effective chemopreventive agents. Thus, development of novel compounds that can target cellular and molecular pathways involved in the multistep carcinogenesis process is urgently needed. Previous studies have suggested that substitution of sulfur by selenium in established cancer chemopreventive agents may result in more effective analogs. Thus in the present study we selected the chemopreventive agent S,S'-(1,4-phenylenebis[1,2-ethanediyl])bisisothiourea (PBIT), also known to inhibit inducible nitric oxide synthase (iNOS), synthesized its selenium analog (Se-PBIT) and compared both compounds in preclinical model systems using non-small cell lung cancer (NSCLC) cell lines (NCI-H460 and A549); NSCLC is the most common histologic type of all lung cancer cases. Se-PBIT was found to be superior to PBIT as an inducer of apoptosis and inhibitor of cell growth. Se-PBIT arrested cell cycles at G1 and G2-M stage in both A549 and H460 cell lines. Although both compounds are weakly but equally effective inhibitors of iNOS protein expression and activity, only Se-PBIT significantly enhanced the levels of p53, p38, p27 and p21 protein expression, reduced levels of phospholipase A2 (PLA2) but had no effect on cyclooxygenase-2 (COX-2) protein levels; such molecular targets are involved in cell growth inhibition, induction of apoptosis and cell cycle regulation. The results indicate that Se-PBIT altered molecular targets that are involved in the development of human lung cancer. Although, the mechanisms that can fully account for these effects remain to be determined, the results are encouraging to further evaluate the chemopreventive efficacy of Se-PBIT against the development of NSCLC in a well-defined animal model.
The Toxic Oil Syndrome (TOS) was a toxic epidemic disease, related to the consumption of rapeseed oil denatured with aniline that affected more than 20,000 people in Spain and resulted in more than 330 deaths after its sudden appearance in 1981. It has been reported that the fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have shown a strong association with TOS. These PAP-esters could be absorbed and metabolized in a similar way than phospholipids. This is of interest because some products of phospholipid metabolism are important mediators in downstream pathways involved in the regulation of different nuclear factors. In particular, phospholipase D activity is involved in the activation of c-fos. Thus, we have investigated the effect of different PAP-esters in the induction of c-fos in lung fibroblasts. Results indicate that PAP-esters rapidly induced the expression of c-fos in a dose-dependent manner. In addition, both butanol and propranolol prevent this induction pointing to the involvement of phospholipase D in this activation. These results suggest that deregulation of some nuclear factors such as AP-1 could be involved in the pathogenesis of TOS.
1,2-Naphthoquinone (1,2-NQ) is an atmospheric electrophile that reacts covalently with protein thiols. Our previous study revealed that exposure of bovine aortic endothelial cells to 1,2-NQ causes covalent modification of cAMP response element-binding protein (CREB), thereby inhibiting its DNA binding activity and substantial gene expression of B-cell lymphoma-2 (Bcl-2) that is regulated by this transcription factor. In this study, we identified the modification sites of CREB that are associated with the decreased transcriptional activity. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis indicated that three amino acids (Cys-286, Lys-290, and Lys-319) were irreversibly modified by 1,2-NQ. Mutational analysis revealed that electrophilic modification of Cys-286, but not the other two amino acids, at the DNA binding domain is essential for the reduced CREB activity. Substitution of Cys-286 with tryptophan (C286W), which mimics CREB modification by 1,2-NQ, supported this notion. These results suggest that the covalent interaction of CREB with 1,2-NQ through Cys-286 blocks the DNA binding activity of CREB, resulting in the repression of CREB-regulated genes.
The similarity between the induction of presumptive preneoplastic hepatocytes by chemicals and cell transformation by radiation and viruses is noteworthy, in that they both require at least one round of cell proliferation. The observed lack of strict, quantitative correlations between rate of liver cell proliferation and induction of liver cell tumours may be in part due to the fact that the end point in these studies was the development of tumours, a process consisting of many steps, several of which and in particular the rate limiting ones have not yet been identified.
Colorectal cancer is the second leading cause of cancer death worldwide with diet playing a prominent role in disease initiation and progression. Diet and nutrition play an important role during this multistep colon carcinogenic process. We have investigated the modulatory efficacy of hesperetin on aberrant crypt foci (ACF) and xenobiotic-metabolizing enzymes on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control, received modified pellet diet and group 2 rats received 20mg/kg body weight of hesperetin p.o. every day. Groups 3-6 rats were given subcutaneous injections of 1,2-dimethylhydrazine (20mg/kg body weight) once a week for 15 weeks to induce ACF in the colon. In addition, rats in group 4 received hesperetin as in group 2 orally for the first 15 weeks (initiation), group 5 rats received hesperetin as in group 2 after the last injection of DMH and continued till the end of the experimental period (post-initiation). Group 6 received hesperetin as in group 2 throughout the entire period of 32 weeks. DMH exposure showed high incidence (90%) of ACF (280+/-24.5 aberrant crypt/colon) and dysplastic ACF, elevated activities of phase I enzymes and reduced the activities of phase II enzymes in the liver and colonic mucosa of colon cancer bearing rats. Hesperetin supplementation significantly reversed these effects, the effect being more pronounced in group 6 rats (hesperetin supplemented throughout the study period). These findings suggest that hesperetin can significantly reduce the formation of preneoplastic lesions and effectively modulate the xenobiotic-metabolizing enzymes in rats.
We studied metabolism of monochlorobenzene (MCB), 1,2-dichlorobenzene (1,2-DCB) and 1,4-DCB in liver microsomes from untreated male and female Wistar rats and B6C3F1 mice or in those after the induction of CYP3A or 2E1 as well as in human male liver microsomes. MCB and 1,2-DCB were oxidised mainly by rat and human CYP2E1. It was found that 1,4-DCB was oxidised by rat and human CYP2E1 at a several-fold lower rate than 1,2-DCB, but a greater part to covalently binding products. In contrast to previous studies showing rat CYP3A1 as the main CYP form oxidising both DCBs, our experiments indicate only a certain role of rat and human CYP3A in MCB, 1,2-DCB and 1,4-DCB oxidation to covalently bound products. The relative roles of human liver CYP2E1 and 3A4 in the metabolism of 1,4-DCB seem to be individually different. Metabolic rates of MCB, 1,2-DCB and 1,4-DCB correlated with CYP2E1 immunochemical level in microsomes from 11 different human livers and with metabolic rates of CYP2E1 substrates. These rates in different human livers were up to 10-fold different and were generally several-fold higher than those in untreated rats or mice. Metabolic activation of MCB and 1,2-DCB to products binding covalently to microsomal proteins and to calf-thymus DNA, respectively, mostly corresponded to production of water-soluble metabolites. Significant species and sex differences in the oxidation of MCB, 1,2-DCB and 1,4-DCB were reflected in a markedly higher oxidation in male mice than male rats and higher oxidation in male than female mice. The formation of covalently bound products generally corresponded to production of soluble metabolites, but female rats formed significantly less covalently bound products of 1,4-DCB (and also of 1,2-DCB and MCB) than male rats and mice of both sexes, in possible reflection of the fact that 1,4-DCB is not carcinogenic in female rats despite its carcinogenicity for male rats and both sexes of mice.
1,3-Butadiene is metabolized mainly by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3-butene (EB), a reactive metabolite. It has been shown that P450s can be inactivated by covalent binding of reactive metabolites to protein or heme. Molecular dosimetry studies have clearly shown that BD metabolism follows a supralinear dose response, suggestive of saturation of metabolic activation. In this study, potential binding sites of EB in human P450 2E1 were identified and modeled to test whether EB covalently binds to residues important for enzyme activity. Commercially available human P450 2E1 was reacted with EB, digested with trypsin and the resulting peptides were analyzed by Matrix-Assisted Laser Desorption/Ionization tandem Time-of-Flight mass spectrometry (MALDI-MS). The identity of EB modified peptides was confirmed by Matrix-Assisted Laser Desorption/Ionization tandem mass spectrometry (MALDI-MS/MS) sequencing. It was shown that EB binds to four histidine and two tyrosine residues. All modification sites were assigned by at least two adjacent and a minimum of eight peptide specific fragments. Protein modeling revealed that two of these covalent modifications (His(109), His(370)) are clearly associated with the active site, and that their Calpha atoms are located less than 9A from a known inhibitor binding site. In addition, the side chain of His(370) is within 4A of the heme group and its modification is expected to influence the orientation of the heme. The Calpha atom of Tyr(71) is within 14A of the potential inhibitor binding site and within 7A of the flap undergoing conformational change upon ligand binding, potentially placing Tyr(71) near the substrate as it enters and leaves the active site. The data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory mechanism for BD metabolism.
The cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP) is active as a result of its bifunctional reactions with DNA. Many other platinum complexes also have therapeutic activity. Of current interest are complexes containing 1,2-diaminocyclohexane (DACH). The DACH ligand exists in three isomeric forms with reported differences in therapeutic activity in the order R,R greater than S,S greater than R,S-DACH-Pt. The reaction of the sulphate form of each of these three isomers with DNA has been characterized as a possible explanation for the apparent differences in antitumor activity. These reactions have been characterized by platinating pure DNA followed by enzyme digestion, HPLC separation and analysis by atomic absorption and nuclear magnetic resonance. The spectrum of adducts produced was similar for each isomer and similar to that reported for cis-DDP with adduction at d(GpG), d(ApG) and (dG)2. The R,S-isomer additionally demonstrated isomeric adducts at d(GpG) and d(ApG). The kinetics of formation of the various adducts was the same for each isomer; total platination of DNA was complete in 15 min as were bifunctional adducts at d(GpG) and (dG)2. However, rearrangement to bifunctional adducts took several hours in the case of adducts at d(ApG) sequences. These results did not provide a reason for the different activities of the isomers. It is suggested that the interaction of these adducts with metabolic processes such as DNA repair might explain these differences.
Studies were performed to determine if the detoxification pathway of 1,3-butadiene (BD) through 3-butene-1,2-diol (BD-diol) is a major contributor to mutagenicity in BD-exposed mice and rats. First, female and male mice and rats (4-5 weeks old) were exposed by nose-only for 6h to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure concentrations that yielded similar plasma levels of BD-diol, and then animals were exposed in inhalation chambers for 4 weeks to BD-diol to determine the mutagenic potency estimates for the same exposure levels and to compare these estimates to those reported for BD-exposed female mice and rats where comparable blood levels of BD-diol were achieved. Measurements of plasma levels of BD-diol (via GC/MS methodology) showed that (i) BD-diol accumulated in a sub-linear fashion during single 6-h exposures to >200 ppm BD; (ii) BD-diol accumulated in a linear fashion during single or repeated exposures to 6-18 ppm BD and then in a sub-linear fashion with increasing levels of BD-diol exposure; and (iii) exposures of mice and rats to 18 ppm BD-diol were equivalent to those produced by 200 ppm BD exposures (with exposures to 36 ppm BD-diol yielding plasma levels approximately 25% of those produced by 625 ppm BD exposures). Measurements of Hprt mutant frequencies (via the T cell cloning assay) showed that repeated exposures to 18 and 36 ppm BD-diol were significantly mutagenic in mice and rats. The resulting data indicated that BD-diol derived metabolites (especially, 1,2-dihydroxy-3,4-epoxybutane) have a narrow range of mutagenic effects confined to high-level BD (>or=200 ppm) exposures, and are responsible for nearly all of the mutagenic response in the rat and for a substantial portion of the mutagenic response in the mouse following high-level BD exposures.
In vitro bromide release and in vivo glutathione (GSH) depletion in rat liver, kidney and testis by 1,2-dibromo-3-chloropropane (DBCP) and selectively methylated and deuterated DBCP analogs were studied. With liver microsomes from phenobarbital-pretreated rats the bromide release from the C1-C3-D4- and the perdeuterated DBCP analogs were 54% and 26% of that of DBCP, respectively. Inhibitors of P-450 reduced the bromide release to 10-20% of that without additions. This correlated with the effects of deuterium substitution and additions of P-450 inhibitors on DBCP-induced bacterial mutagenicity as reported elsewhere by this laboratory. To study the importance of GSH-dependent metabolism in DBCP toxicity, bromide release was assayed in cytosolic preparations using methylated analogs of DBCP. With the C1-methyl-derivative, bromide release was markedly reduced compared to that with DBCP in cytosols from liver, kidney and testis. A similar reduction in in vivo nephrotoxicity and testicular damage has recently been reported. The obtained correlation between in vitro GSH-dependent metabolism of methylated DBCP analogs and their in vivo organ damaging potential, points to an involvement of GSH-dependent metabolism in DBCP-induced in vivo toxicity. Both DBCP and the methylated analogs (360 mumol/kg i.p.) depleted the GSH levels in liver after 1 and 3 h and in kidney after 1 h, whereas in the testis no significant depletion of GSH was obtained. As kidney and testis are reported to be the primary target organs for DBCP, there was an apparent lack of correlation between tissue depletion of GSH and organ toxicity.
The microsomal oxidation of 1,2-[14C]- and 1,4-[14C]dichlorobenzene (DICB) was investigated with special attention for possible differences in biotransformation that might contribute to the isomer-specific hepatotoxicity. Major metabolites of both isomers were dichlorophenols (2,5-DICP for 1,4-DICB and 2,3- and 3,4-DICP for 1,2-DICB, respectively) and dichlorohydroquinones. The formation of polar dihydrodiols appeared to be a major route for 1,2-DICB but not 1,4-DICB. Both the hepatotoxic 1,2-DICB and the non-hepatotoxic 1,4-DICB were oxidized to metabolites that covalently interacted with protein and only to a small extent with DNA. Protein binding could be inhibited by the addition of the reducing agent ascorbic acid with a concomitant increase in the formation of hydroquinones and catechols, indicating the involvement of reactive benzoquinone metabolites in protein binding. However, in the presence of ascorbic acid, a substantial amount of protein-bound metabolites of 1,2-DICB was still observed, in contrast to 1,4-DICB where binding was nearly completely inhibited. This latter effect was ascribed to the direct formation of reactive benzoquinone metabolites in a single P450-mediated oxidation of para-substituted dichlorophenols (such as 3,4-DICP) in the case of 1,2-DICB. In contrast, the major phenol isomer derived from 1,4-DICB (i.e. 2,5-DICP) is oxidized to its hydroquinone derivative, which needs prior oxidation in order to generate the reactive benzoquinone species. Residual protein binding in the presence of ascorbic acid could also indicate the involvement of reactive arene oxides in the protein binding of 1,2-DICB, but not of 1,4-DICB. However, MO computer calculations did not provide indications for differences in chemical reactivity and/or stability of the various arene oxide/oxepin tautomers that can be formed from either 1,2-DICB or 1,4-DICB. In conclusion, reactive intermediates in the secondary metabolism of 1,2-DICB lead to more covalent binding than those derived from 1,4-DICB, which correlates very well with their reported hepatotoxic potency.
The carcinogenicity of 1,3-butadiene (BD) is related to its bioactivation to several DNA-reactive metabolites; accumulating evidence suggests that the stereochemistry of these BD intermediates may play a significant role in the mutagenic and carcinogenic actions of the parent compound. The objective of this study was to evaluate the cytotoxicity and mutagenicity of stereochemical forms of 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB), two genotoxic BD metabolites, in a human lymphoblastoid cell line, TK6. Cytotoxicity was measured by comparing cloning efficiencies in chemical-exposed cells versus those in control cells. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) and thymidine kinase (TK) mutant frequencies (MFs) were measured using a cell cloning assay. HPRT mutants collected from cells exposed to the three forms of DEB were analyzed by PCR to characterize large genetic alterations. All the three stereoisomers of DEB caused increased HPRT and TK MFs compared to the concurrent control samples. There were no significant differences in cytotoxicity or mutagenicity among the three isomers of DEB in TK6 cells. Molecular analysis of HPRT mutants revealed similar distributions of types of mutations among the three isomers of DEB. There were also no statistically significant differences in mutagenic efficiencies between the two isomers of EB in TK6 cells. These results were consistent with the in vivo findings that there was little difference in the mutagenic efficiencies of racemic-DEB versus meso-DEB in rodents. Thus, in terms of mutagenic efficiency, stereochemical configurations of EB and DEB are not likely to play a significant role in the mutagenicity and carcinogenicity of BD.
In the rat, a single ethanol (EtOH) pretreatment (2.5 g/kg b.w., per os) was able to strongly enhance the cytotoxicity of 1,2-dibromoethane (DBE)(87 mg/kg b.w., per os). The principal metabolic routes of DBE involve both oxidative and conjugative transformations. Microsomal cytochrome P450 content and dimethyl nitrosamine demethylase activity were not changed, while a significant loss of cytosolic total GSH-transferase was observed in rats killed 6 h after EtOH pretreatment. Pretreatment with methylpyrazole, an inhibitor of alcohol-dehydrogenase prevented the effects provoked by ethanol. The major EtOH metabolite, acetaldehyde. seemed thus to play a fundamental role in the mechanism responsible for the potentiation of DBE toxicity mediated by EtOH. To further support this hypothesis, disulfiram (75 mg/kg b.w.), an inhibitor of aldehyde dehydrogenase, was given i.p. to rats. When DBE was administered to disulfiram- and EtOH-pretreated rats, a marked increase of liver cytolysis was shown and cytosolic GSH-transferase activity was further inhibited if compared to that induced by EtOH treatment alone. The results are consistent with the hypothesis that EtOH given to rats increases DBE liver toxicity because its major metabolite, acetaldehyde, reduces the DBE conjugates to GSH transferase, with consequent shift of DBE metabolism to the oxidative route and accumulation of reactive oxidative intermediates no longer effectively conjugated with GSH.
Oltipraz is a cancer chemopreventive agent active against a wide variety of chemical carcinogens. In spite of the intense chemoprevention and toxicology studies on oltipraz, no information is available on its antifibrotic efficacy. In the present study, the effects of oltipraz on dimethylnitrosamine (DMN)-induced liver fibrogenesis were assessed in rats. As part of mechanistic studies, the expression of transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha) was monitored. Treatment of rats with DMN (10 microl/kg body weight, i.p., three times per week for 4 weeks) resulted in marked increases in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transpeptidase (gamma-GT) activities. DMN also caused an increase in the plasma bilirubin content, whereas total plasma protein and albumin levels were rather decreased. Oltipraz (50 mg/kg body weight, p.o., three times per week for 4 weeks) inhibited the increases in plasma ALT, AST, gamma-GT and bilirubin by DMN. DMN increased liver fibrosis as histopathologically assessed by Van Gieson's staining and Masson's trichrome staining (fibrosis score, 3.7; Knodell score, 16), which was reduced by oltipraz treatment (fibrosis score, 2.5; Knodell score, 8.0). Reverse transcription-polymerase chain reaction analysis revealed that oltipraz inhibited an increase in the TGF-beta1 mRNA by DMN. Oltipraz was also active in reducing the production of plasma TNF-alpha by DMN or lipopolysaccharide (LPS), which would contribute to its cytoprotective effect. These results demonstrated that oltipraz inhibited hepatocyte injury and impairment of liver function induced by DMN, and reduces DMN-induced liver fibrosis possibly through suppression of TGF-beta1 and TNF-alpha production.
Colorectal cancer is one of the most common internal malignancies in Western society. Currently oxidative stress has been increasingly postulated as a major contributor to carcinogenesis. The assessment of damage in various biological matrices, such as tissues and cells, is vital to understand the development of carcinogenesis and subsequently devising intervention strategies. Thus, the major objective of the present study was to examine the effect of resveratrol (Res) on DNA damage in a short-term study of 16 days and circulatory lipid peroxidation, enzymic/non-enzymic antioxidants status in a long-term study of 30 weeks in 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis. Wistar male rats were divided into 6 groups, group 1 were control rats, group 2 rats received Res (8mg/kg body weight, orally, everyday), rats in groups 3-6 were administered (DMH, 20mg/kg body weight, s.c.) as four injections in order to induce DNA damage in the short-term or once a week for the first 15 weeks in the long-term study. In addition to DMH, group 4 (initiation), 5 (post-initiation) and 6 (entire-period) received Res (8mg/kg body weight, p.o., everyday). The results revealed that, supplementation with Res (entire-period) treatment regimen significantly reduced the DMH-induced leukocytic DNA damage (tail length, tail moment, % DNA in the comet tail and olive tail moment) as compared to DMH-alone treated rats. In addition, entire-period Res supplementation increased the enzymic (superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase and glutathione S-transferase) and non-enzymic (reduced glutathione, vitamin C, vitamin E and beta-carotene) antioxidant status with a corresponding decrease in the extent of lipid peroxidation markers (thiobarbituric acid reactive substances, diene conjugates and lipid hydroperoxides). Conversely, Res supplementation during initiation and post-initiation regimen did not produce greater modulatory effects. Our results indicate that DMH-induced DNA damage and oxidative stress were suppressed/prevented effectively by chronic Res supplementation.