Rat Liver S9 Fraction Research Articles

Characterization of antimutagenic mechanism of 3-allyl-5-substituted 2-thiohydantoins against 2-amino-3-methylimidazo[4,5- f]quinoline

3-Allyl-5-substituted 2-thiohydantoins (ATH-amino acids) derived from allyl isothiocyanate and amino acids can inhibit the mutagenicity of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) in the Salmonella assay. In this report, we studied possible mechanisms for the inhibition using rat liver S9 in assays for ethoxyresorufin O-deethylase (EROD), a marker activity for cytochrome P450 1A (CYP1A), which activates heterocyclic amines, and the Salmonella assays with the direct-acting mutagen 2-hydroxyamino-3-methylimidazo[4,5- f]quinoline ( N-hydroxy-IQ). Quantitative analysis of ATH-amino acids and IQ during incubation with rat liver S9 fraction by HPLC showed that ATH-amino acids could act as S9-inhibitors, thereby inhibiting metabolic activation of IQ. Among the tested ATH-amino acids, ATH-Phe, ATH-Trp, ATH-Leu and ATH-Val showed a dose-dependent inhibition of EROD activity. ATH-Gly, ATH-Glu, and ATH-Asp behaved as blocking agents toward N-hydroxy-IQ, but exhibited no inhibition of EROD activity.

Evaluation of β-myrcene, α-terpinene and (+)- and (−)-α-pinene in the Salmonella/microsome assay

This study was undertaken to investigate the genotoxicity of β-myrcene, α-terpinene and (+) and (−)-α-pinene, monoterpenes found in a variety of plant volatile oils. β-myrcene, α-terpinene and α-pinene as well as plant oils containing these hydrocarbon monoterpenes have been used as flavoring additives in foods and beverages, as fragrances in cosmetics, and as scent in household products. Mutagenicity was evaluated by the Salmonella/microsome assay (TA100, TA98, TA97a and TA1535 tester strains), without and with addition of an extrinsic metabolic activation system (rat liver S9 fraction induced by Aroclor 1254). Two dose-complementary assays were performed so that a broad range of doses, including a number of regularly-spaced doses in the non-toxic dose interval, were tested. No increase in the number of his+ revertant colonies over the negative control values was observed in any of the four S. typhimurium tester strains. Results from the present study therefore indicated that β-myrcene, α-terpinene, and (+) and (−)-α-pinene are not mutagenic in the Ames test.

Lack of DNA damage induction by okadaic acid, a marine toxin, in the CHO-Hprt and the in vitro UDS assays

Okadaic acid (OA) is a marine toxin produced by dinoflagellates and responsible for human intoxications. OA is a specific inhibitor of serine/threonine protein phosphatases PP1 and PP2A and a potent tumor promoter in mouse skin and rat glandular stomach. In a previous study, we demonstrated that OA induced aneuploidy in CHO-K1 cells using the cytokinesis-block micronucleus (CBMN) assay coupled to FISH and concluded that OA was not a direct mutagen. As some previous in vitro mutagenicity studies had given positive results with OA, we decided to perform two additional in vitro mutagenicity assays in accordance with the OECD guidelines: (i) the CHO/Hprt test, which provides end points about locus-specific gene mutation; (ii) the in vitro unscheduled DNA synthesis (UDS) assay in rat hepatocytes, which measures [ 3H]thymidine incorporation into DNA undergoing excision repair. In the CHO/Hprt assay, there was no significant increase in the number of mutants for doses ranging from 5 to 5000 nM in the presence or absence of rat liver S9 fraction. In the in vitro UDS assay, OA did not induce primary DNA damages in rat hepatocytes following 18 h exposure at concentrations between 1.32 and 100 nM. As OA could affect the DNA repair systems via the inhibition of protein phosphatases, its effects on the repair kinetic of 2AAF-induced DNA damage were also investigated with the UDS assay. The results showed that OA did not interact with the DNA-repair process involved in in vitro UDS in rat hepatocytes. We concluded that OA failed to induce direct DNA damage but acted principally by altering the chromosome number, which could contribute to its carcinogenic effect.

Expression and activities of several drug-metabolizing enzymes in LLC-PK1 cells

LLC-PK1 cells are frequently used in toxicology research, but little information is available concerning the capacity of these cells to metabolize xenobiotics. We examined the expression and activities of cytochromes P450 (P450) 1A1/1A2 (CYP 1A1/1A2), 2E1 (CYP 2E1), flavin monooxygenase (FMO), 5-lipoxygenase (5-LO) and prostaglandin H synthase (PHS)-associated cyclooxygenase-1 (COX-1). We prepared S9 fractions from LLC-PK1 cells, rat liver, and rat kidney, and measured enzyme activities using ethoxyresorufin O-deethylation (EROD) for CYP 1A1/1A2 and ethoxycoumarin O-deethylation (ECOD) for CYP 2E1, benzydamine N-oxidation (BNO) for FMO, leukotriene B 4 (LTB 4) formation for 5-LO, and thromboxane B 2 (TXB 2) formation for COX-1 activities. To assure that product formation was due to enzymatic activity, we used the following inhibitors: 1-aminobenzotriazole (ABT) for P450, methimazole for FMO, caffeic acid for 5-LO and acetylsalicylic acid (ASA) for COX-1. We also performed Western blot analysis to confirm our observations. All five enzyme activities were demonstrable in rat liver at much greater levels than in rat kidney S9 fractions. Activities in LLC-PK1 cells were significantly lower than activities in rat liver S9 fraction and generally less than activities in rat kidney S9 fraction. Enzyme inhibitors decreased product formation in all three tissues and Western blot analysis supported our observations of low enzyme activity in LLC-PK1 cells. These results indicate that LLC-PK1 cells have very low content of relevant drug-metabolizing enzyme activities.

In vivo mutagenicity of benzo[f]quinoline, benzo[h]quinoline, and 1,7-phenanthroline using the lacZ transgenic mice

Phenanthrene, a simplest angular polycyclic aromatic hydrocarbon with a bay-region in its molecule, is reported to be non-mutagenic, although most angular (non-linear) polycyclic aromatic hydrocarbons, such as benzo[a]pyrene and chrysene, are known to show genotoxicity after metabolic transformation into a bay-region diol epoxide. On the other hand, benzo[f]quinoline (BfQ), benzo[h]quinoline (BhQ), and 1,7-phenanthroline (1,7-Phe), which are all aza-analogs of phenanthrene, are mutagenic in the Ames test using Salmonella typhimurium TA100 in the presence of a rat liver S9 fraction. In this report, we undertook to investigate the in vivo mutagenicity of BfQ, BhQ and 1,7-Phe by an in vivo mutation assay system using the lacZ transgenic mouse (Muta™Mouse). BfQ and BhQ only slightly induced mutation in the liver and lung, respectively. BfQ- and BhQ-induced cII mutant spectra showed no characteristics compared with that of the control. These results suggest that the in vivo mutagenicities of BfQ and BhQ were equivocal. On the other hand, 1,7-Phe induced a potent mutation in the liver and a weak mutation in the lung. Furthermore 1,7-Phe depressed the G:C to A:T transition and increased the G:C to C:G transversion in the liver like quinoline, a hepatomutagen possessing the partial structure of 1,7-Phe, compared with the spontaneous mutation spectrum. These results suggest that the in vivo mutagenicity of 1,7-Phe might be caused by the same mechanism as that of quinoline, which induced the same mutational spectrum change (G:C to C:G transversion).

Simultaneous quantitative analysis of three drugs by high‐performance liquid chromatography/electrospray ionization mass spectrometry and its application to cassette in vitro metabolic stability studies

A sensitive and selective liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) method has been developed for the simultaneous quantitative determination of three new chemical entities (NCEs), of the class of aryloxy-substituted aryl piperazines, in rat liver S9 fraction. S9 fraction samples (0.1 mL) were simply extracted with 2% isopropanol in diethyl ether and the extracts analyzed by HPLC with the detection of the analytes in the selective ion recording (SIR) mode. The determination of the analytes was accurate and reproducible, with a limit of quantification of 50 ng/mL for all the analytes in rat liver S9 fraction. The standard calibration curve for the analytes was linear over the concentration range 50-4000 ng/mL. Analysis accuracy and precision over the concentration range were lower than +/-15%. This method offered significant increase in the analytical throughput, which is illustrated by the 'N-in-One' study of metabolic stability of the compounds in rat liver S9 fractions. The quantitative results from the 'N-in-One' procedure correlated well with those obtained from conventional discrete analyses. In addition, the samples were reanalyzed to allow for detection of the metabolites formed during the same incubation. The metabolites were first characterized by nominal mass measurement of the corresponding protonated molecules. Subsequent tandem mass spectrometry allowed confirmation of the detected metabolites.

Liquid chromatography–electrospray ionization mass spectrometry for metabolism and pharmacokinetic studies of ginsenoside Rg3

A sensitive and specific liquid chromatography (LC)–electrospray ionization (ESI) mass spectrometry (MS) method with quadrupole-time-of-flight detection was applied for the determination of ginsenoside Rg3 in rat plasma and its major metabolites in samples from in vitro metabolism studies. The biological samples were prepared by protein precipitation with methanol and the sample extracts were directly injected into a reversed-phase HPLC column with solvent gradient program. Pharmacokinetic study of Rg3 on rat gave a half-life of 14 min after the ginsenoside was intravenously dosed. Metabolite identification and confirmation from the MS–MS and high-resolution MS analyses indicated that the short half-life might be due to the high rate of metabolic clearance of the ginsenoside under gastrointestinal conditions. Hydrolysis products of Rg3 such as ginsenoside Rh2 and protopanaxadiol were identified as the major metabolites from the incubation with 0.1 M HCl, and a hydrated product of protopanaxadiol was detected as the metabolite with artificial gastric juice, while a monooxygenated metabolite was identified in the incubated samples with rat liver S9 fraction.

Deviation from additivity in mixture toxicity: relevance of nonlinear dose-response relationships and cell line differences in genotoxicity assays with combinations of chemical mutagens and gamma-radiation.

Sublinear dose-response relationships are often seen in toxicity testing, particularly with bioassays for carcinogenicity. This is the result of a superimposition of various effects that modulate and contribute to the process of cancer formation. Examples are saturation of detoxification pathways or DNA repair with increasing dose, or regenerative hyperplasia and indirect DNA damage as a consequence of high-dose cytotoxicity and cell death. The response to a combination treatment can appear to be supra-additive, although it is in fact dose-additive along a sublinear dose-response curve for the single agents. Because environmental exposure of humans is usually in a low-dose range and deviation from linearity is less likely at the low-dose end, combination effects should be tested at the lowest observable effect levels (LOEL) of the components. This principle has been applied to combinations of genotoxic agents in various cellular models. For statistical analysis, all experiments were analyzed for deviation from additivity with an n-factor analysis of variance with an interaction term, n being the number of components tested in combination. Benzo[a]pyrene, benz[a]anthracene, and dibenz[a,c]anthracene were tested at the LOEL, separately and in combination, for the induction of revertants in the Ames test, using Salmonella typhimurium TA100 and rat liver S9 fraction. Combined treatment produced no deviation from additivity. The induction of micronuclei in vitro was investigated with ionizing radiation from a 137Cs source and ethyl methanesulfonate. Mouse lymphoma L5178Y cells revealed a significant 40% supra-additive combination effect in an experiment based on three independent replicates for controls and single and combination treatments. On the other hand, two human lymphoblastoid cell lines (TK6 and WTK1) as well as a pilot study with human primary fibroblasts from fetal lung did not show deviation from additivity. Data derived from one cell line should therefore not be generalized. Regarding the testing of mixtures for deviation from additive toxicity, the suggested experimental protocol is easily followed by toxicologists.

Isolation and characterization of metabolites of centpropazine in rat liver, intestine, and red blood cell homogenates

The potential sites for metabolism of centpropazine (CPZ) (an antidepressant) were evaluated in male Sprague‐Dawley rats. The isolation and identification of the major metabolites formed in the presence of rat liver S9 fraction, intestine, and red blood cells under aerobic conditions were performed using high‐performance liquid chromatography and electrospray ionization mass spectrometry. CPZ was found to be extensively metabolized to seven possible metabolites by liver S9 fraction in the presence of a nicotinamide adenine dinucleotide phosphate generating system at 37°C. Both intestinal wall and red blood cells were also found to metabolize the compound. This metabolite structure was confirmed by comparison with that of its synthetic standard. The drug was stable in intestinal contents. On the basis of our finding, we propose the in vitro metabolic pathways for CPZ. © 2002 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2067–2075, 2002

Synthesis, in vitro metabolism, cell transformation, mutagenicity, and DNA adduction of dibenzo[c,mno]chrysene.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants. Due to its structural similarity with the potent carcinogen dibenzo[a,l]pyrene (DB[a,l]P) and because of its environmental presence, dibenzo[c,mno]chrysene (naphtho[1,2-a]pyrene, N[1,2-a]P) is of considerable research interest. We therefore developed an efficient synthesis of N[1,2-a]P, and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction. Its mutagenic activity in S. typhimurium TA 100 and its morphological cell transforming ability in mouse embryo fibroblasts were evaluated. On the basis of spectral analyses, the in vitro major metabolites were identified as the fjord region dihydrodiol trans-9,10-dihydroxy-9,10-dihydro-N[1,2-a]P (N[1,2-a]P-9,10-dihydrodiol), the K-region diols N[1,2-a]P-4,5-dihydrodiol and N[1,2-a]P-7,8-dihydrodiol, and also the 1-, 3-, and 10-hydroxy-N[1,2-a]P; the structure of N[1,2-a]P-9,10-dihydrodiol was also confirmed by independent synthesis. In assays with S. typhimurium TA 100, N[1,2-a]P-9,10-dihydrodiol was half as mutagenic as (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) at > or =4 nmol/plate. N[1,2-a]P-9,10-dihydrodiol was much more mutagenic than N[1,2-a]P at all dose levels, suggesting that the N[1,2-a]P-9,10-dihydrodiol is the likely proximate mutagen of N[1,2-a]P. Evaluation of morphological cell transformation in C3H10T1/2C18 mouse embryo fibroblasts revealed that N[1,2-a]P was comparable to B[a]P. We further examined the pattern of in vitro adduct formation between calf thymus DNA and (+/-)-anti-9,10-dihydroxy-9,10-dihydro-11,12-epoxy-9,10,11,12-tetrahydro-N[1,2-a]P (N[1,2-a]PDE) and found that dG-adduct formation is 2.9-fold greater than dA-adduct formation. On the basis of our results and those reported in the literature, our working hypothesis is that N[1,2-a]P may be added to the list of potent carcinogens that includes DB[a,l]P. This hypothesis is currently being tested in our laboratory.

Mutagenicity of steviol and its oxidative derivatives in Salmonella typhimurium TM677.

Stevioside is natural non-caloric sweetner isolated from Stevia rebaudiana BERTONI, which has been used as a non-caloric sugar substitute in Japan. Pezzuto et al. demonstrated that steviol shows a dose-dependent positive response in forward mutation assay using Salmonella typhimurium TM677 in the presence of metabolic activation system (Aroclor induced rat liver S9 fraction). Our studies were carried out to identify the genuine mutagenic active substance from among the eight steviol derivatives. Steviol indicate almost similar levels of mutagenicity under the presence of S9 mixture, as reported by Pezzuto et al. 15-Oxo-steviol was found to be mutagenic at the one tenth the level of steviol itself under the presence of S9 mixture. Interestingly, specific mutagenicity of the lactone derivative under the presence of S9 mixture was ten times lower than that of the lactone derivative without the addition of S9 mixture.

Metabolic activation of bisphenol A by rat liver S9 fraction.

Bisphenol A (BPA) is a well-known endocrine-disrupting chemical found in the environment. To assess the metabolic modulation of estrogenic activity of BPA after ingestion, we investigated whether the incubation of BPA with rat liver S9 fraction results in metabolic activation or inactivation of estrogenic activity using a recombinant yeast expressing human estrogen receptor and MCF-7 transfected firefly luciferase plasmid for a reporter assay. When 0.1 mM BPA was incubated with rat liver S9 for 1 h, the estrogenic activity was increased about two to five times compared with that of the control, in which the S9 was inactivated prior to incubation. This metabolic activation was inhibited by SKF 525-A, an inhibitor of cytochrome P450. With increasing incubation time, the estrogenic activity increased time-dependently. Interestingly, however, the metabolic activation did not proceed with either microsomes or cytosol alone and was restored by a recombination of both fractions. The active metabolite was eluted at later retention time than that of BPA on HPLC with a reversed-phase column. Bisphenol B and methoxychlor were also activated by incubation with rat liver S9, whereas 4-tert-octylphenol and 4-nonylphenol, as well as 17beta-estradiol, were metabolically inactivated. The present results clearly indicate that BPA is metabolically activated in terms of estrogenicity under the conditions existing only with combined rat liver microsomes and cytosol.

Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay.

The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was approximately 10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.

Mutagenicity of N-nitrosodiethylamine in the Ames test with S. typhimurium TA1535 is due to volatile metabolites and is not dependent on cytochrome P4502E1 induction.

N-Nitrosodiethylamine (NDEA) is carcinogenic in all investigated animal species at relatively low dosages. No threshold has been detected for these carcinogenic effects. The substance has been extensively investigated in various in vitro systems, revealing only weak mutagenicity at relatively high dosages. We reinvestigated NDEA in the Ames test with Salmonella typhimurium TA1535 to establish appropriate modifications of the standard Ames test protocol, to achieve a dose-dependent mutagenic response at a reasonably low dose range. Two main modifications were evaluated. Since the metabolism of dialkylnitrosamines is postulated to be mainly dependent on cytochrome P4502E1, a pyrazole-induced rat liver S9 was applied. The second modification involved a gastight preincubation, since metabolites of NDEA might evaporate from the incubation mixture. Cytochrome P4502E1 induction in Wistar rats was achieved by pyrazole treatment. For comparison, a rat liver S9-fraction produced by beta-naphtoflavone/phenobarbital induction was used. N-Nitrosopyrrolidine served as positive control for pyrazole-induced S9-mix with TA1535. NDEA showed no mutagenic response under all test conditions in the presence of pyrazole-induced S9-mix. A strong mutagenic response, exceeding the base rate up to 15-fold at a dose range of 25-1000 microg/plate, was observed using beta-naphtoflavone/phenobarbital-induced S9-mix, gastight preincubation and TA1535. In conclusion the Ames test with gastight preincubation can be useful for the testing of volatile compounds or substances leading to gaseous metabolites. The weak response of NDEA in the Ames test observed previously seems mainly to be due to the volatile character of its mutagenic metabolites. Our results do not support the hypothesis that cytochrome P4502E1 is a major toxifying enzyme for the formation of Ames-test-positive metabolites from NDEA.

Metabolism of dauricine and identification of its main metabolites.

To study the metabolism of dauricine in vivo and in vitro and identify the structure of its main metabolites, urine of rats after drug administration as the samples of dauricine metabolism in vivo was studied. Rat liver S9 fraction was prepared and the oxygenation metabolism system reconstituted to perform phase I reaction of dauricine in vitro. TLC, HPLC-DAD and MS were used to analyze and identify dauricine and its main phase I metabolites in the samples. The results showed that besides the untransformed dauricine, in the urine samples there was little product of X' which had the same features of TLC, HPLC-DAD and MS as those of N-desmethyl dauricine (N-ddau). Part of dauricine could be transformed to a main metabolite X after incubating with S9 fraction in appropriate conditions. The molecular ion peak of X was m/z 611. The full scan MS2 spectrum of m/z 611 peak from S9 sample were m/z 580, m/z 566, m/z 552, m/z 206, which were same as those of N-ddau. Liver is the major organ for dauricine metabolism and part of dauricine is biotransformed by liver. The major metabolite is considered to be N-ddau.

Genetic toxicity of cocaine.

Cocaine is a widely abused drug. Recently, it has been shown to induce teratogenesis in both humans and animals. Cocaine-induced teratogenicity has been associated with reactive oxygen species (ROS), which are generated by cytochrome P450 during cocaine biotransformation. Since ROS have been reported to induce genotoxicity, it is of interest to know whether cocaine and/or its metabolites are also genotoxic. In this study, Chinese hamster ovary K1 cells were employed as a model system to investigate the genetic toxicity of cocaine in the presence or absence of rat liver S9 fraction. Cocaine-induced cytotoxicity was potentiated when S9 was present, indicating the cytochrome P450 metabolism plays a role in cocaine-mediated cytotoxicity. Cocaine treatments per se induced a few chromosome aberrations while treatments of cocaine plus S9 caused a significant increase in chromosome aberrations. In contrast, cocaine induced micronuclei (MN) formation and hypoxanthine-guanine phosphoribosyltransferase mutation only in the presence of S9. Therefore, cocaine itself is at best a weak clastogen, whereas metabolite(s) of cocaine is/are truly inducer(s) of clastogenesis and mutagenesis. Cocaine treatments alone also induced a significant increase in sister chromatid exchange frequency but the addition of S9 did not affect the results. Free radical scavengers, including superoxide dismutase and catalase, efficiently decreased the frequency of cocaine plus S9-induced MN, implying that ROS are indeed important components in cocaine-induced genotoxicity. The observation that non-toxic doses of cocaine can inhibit intercellular metabolic cooperation suggests that cocaine may also be a tumor promoter. Our data supports that cocaine could possess genotoxicity in addition to its well-known neurotoxicity and teratogenicity.

Escherichia coli MTC, a human NADPH P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450 isoforms 1A1, 1A2, 2A6, 3A4, or 3A5: catalytic activities and mutagenicity studies

We report here on the genetic engineering of four new Escherichia coli tester bacteria, coexpressing human CYP1A1, CYP2A6, CYP3A4 or CYP3A5 with human NADPH cytochrome P450 reductase (RED) by a biplasmid coexpression system, recently developed to express human CYP1A2 in the tester strain MTC. The four new strains were compared for CYP- and RED-expression levels and CYP activities with the formerly developed CYP1A2 expressing strain. CYP1A2 and CYP2A6 were expressed at the highest, CYP1A1 at the lowest and CYP3A4 and CYP3A5 at intermediate expression levels. Membranes of all five tester bacteria demonstrated similar RED-expression levels, except for the two CYP3A-containing bacteria which demonstrated slightly increased RED-levels. CYP-activities were determined as ethoxyresorufin deethylase (CYP1A1 and CYP1A2), coumarin 7-hydroxylase (CYP2A6) and erythromycin N-demethylase (CYP3A4 and CYP3A5) activities. Reaction rates were comparable with those obtained previously for these CYP–enzymes, except for CYP3A5 which demonstrated a lower activity. Benzo[ a]pyrene and 7,12-dimethylbenz[ a]anthracene demonstrated mutagenicity in the CYP1A1 expressing strain with mutagenic activities, respectively, approximately 10-fold and 100-fold higher as compared with those obtained with the use of rat liver S9 fraction. Aflatoxin B1 demonstrated a significant mutagenicity with all CYP expressing strains, albeit lower as compared to those obtained with the use of rat liver S9. CYP1A2 was approximately 3-fold more effective in generating a mutagenic response of AFB1 as compared to CYP3A4. CYP3A5 and CYP3A4 demonstrated comparable capacities in AFB1 bioactivation which was equal as found for CYP1A1. It is concluded that these four new strains contain stable CYP- and RED-expression, significant CYP-activities and demonstrated significant bioactivation activities with several diagnostic carcinogens.

Prodrugs for systemic bioreductive activation-independent delivery of phyllohydroquinone, an active form of phylloquinone (vitamin K1). 1: Preparation and in vitro evaluation.

With the aim of overcoming the delivery problems (water-solubility and bioreductive activation problems) of phyllohydroquinone (PKH), an active form of phylloquinone (PK, vitamin K1), the N,N-dimethylglycine esters of phyllohydroquinone (1-mono, 1; 4-mono, 2; and 1,4-bis, 3) have been synthesized and assessed in vitro as a prodrug for the systemic bioreductive activation-independent delivery of PKH. The hydrochloride salts of the esters were found to be quite soluble in water. Hydrolysis of the esters in rat liver S9 fraction, rat plasma and phosphate buffer, pH 7.4, at 37 degrees C, was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in rat liver and rat plasma and quantitatively yielded PKH. The enzymatic cleavage and the vitamin K-dependent carboxylation activity of the esters in the rat liver microsome preparation at pH 7.2 and 25 degrees C were studied. The regeneration of PKH from the esters was catalyzed by carboxylesterases located in the rat liver microsome, and the order was as follows: 1 > 3 > 2. The carboxylation was stimulated by selected ester 1 in the absence of dithiothreitol, an activator of the vitamin K cycle. The carboxylation activity of 1 was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. Compound 1 could also stimulate carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. These results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of PKH are potential candidates for parenteral prodrugs which can thus achieve the systemic bioreductive activation-independent delivery of PKH.

Mutagenic interactions of model chemical mixtures

Although current methodology for human health risk assessment assumes additive interactions among the contaminants of a complex mixture, chemical interactions may occur which produce synergistic or antagonistic effects. In this study, the mutagenic response of three model compounds, benzo(a)pyrene (B(a)P), pentachlorophenol (PCP) and 2,4,6-trinitrotoluene (TNT), were tested individually and in binary and tertiary solutions, using the Salmonella/microsome assay with each of three bacterial tester strains (TA97a, TA98, and TA100). For all strains, B(a)P was mutagenic with metabolic activation (Arochlor 1254-induced Sprague-Dawley rat liver S9 fraction), TNT was mutagenic without metabolic activation, and pentachlorophenol was inactive both with and without metabolic activation. In binary and tertiary solutions, pentachlorophenol had no effect on the mutagenicity of B(a)P or TNT, independent of metabolic activation. For strain TA97a, the mutagenicity of B(a)P with metabolic activation was slightly decreased in the presence of TNT; the mutagenicity of TNT without metabolic activation was slightly decreased in the presence of B(a)P and PCP; and the mutagenicity of the tertiary solution (496 revertants/10 ug) with metabolic activation was lower than the mutagenicity of B(a)P alone (729 revertants/10 ug). The mutagenicity of B(a)P in strain TA98 with activation was inhibited by the addition of TNT. Studies conducted using several concentrations of TNT or B(a)P indicate that the inhibition of B(a)P mutagenicity was increased as the concentration of TNT increased. Assays performed using four concentrations of S9 indicated the inhibition of B(a)P mutagenicity was relatively unaffected by the level of S9. The data suggest that an interaction in the presence of TNT limits the concentration of B(a)P that is capable of reaching or binding with bacterial DNA.

Mutagenicity testing of (±)-camphor, 1,8-cineole, citral, citronellal, (−)-menthol and terpineol with the Salmonella/microsome assay

The essential oils and their monoterpenoid constituents have been widely used as fragrances in cosmetics, as flavouring food additives, as scenting agents in a variety of household products, as active ingredients in some old drugs, and as intermediates in the synthesis of perfume chemicals. The present study was undertaken to investigate the mutagenic potential of six monoterpenoid compounds: two aldehydes (citral and citronellal), a ketone ((±)-camphor), an oxide ( 1,8-cineole, also known as eucalyptol), and two alcohols (terpineol and (−)-menthol). It is part of a more comprehensive toxicological screening of monoterpenes under way at our laboratory. Mutagenicity was evaluated by the Salmonella/microsome assay (TA97a, TA98, TA100 and TA102 tester strains), without and with addition of an extrinsic metabolic activation system (lyophilized rat liver S9 fraction induced by Aroclor 1254). In all cases, the upper limit of the dose interval tested was either the highest non-toxic dose or the lowest dose of the monoterpene toxic to TA100 strain in the preliminary toxicity test. No mutagenic effect was found with (±) camphor, citral, citronellal, 1,8-cineole, and (−)-menthol. Terpineol caused a slight but dose-related increase in the number of his + revertants with TA102 tester strain both without and with addition of S9 mixture. The results from this study therefore suggest that, with the exception of terpineol, the monoterpenoid compounds tested are not mutagenic in the Ames test.