Rat Liver S9 Preparations Research Articles

Effect of structural modification on the gastrointestinal stability and hepatic metabolism of α-aminoxy peptides

α-Aminoxy peptide AxyP1 has been reported to form synthetic chloride channel in living cells, thus it may have therapeutic potential for the treatment of diseases associated with chloride channel dysfunction. However, this study revealed significant gastrointestinal (GI) instability and extensive hepatic metabolism of AxyP1. To improve its GI and metabolic stability, structural modifications were conducted by replacing the isobutyl side chains of AxyP1 with methyl group (AxyP2), hydroxymethyl group (AxyP3), 4-aminobutyl group (AxyP4) and 3-carboxyl propyl group (AxyP5). Compared with AxyP1 (41 and 47 % degradation), GI stability of the modified peptides was significantly improved by 8-fold (AxyP2), 9-fold (AxyP3) and 12-fold (AxyP5) with no degradation for AxyP4 in simulated gastric fluid within 1 h, and by 12-fold (AxyP2) and 9-fold (AxyP3) with no degradation for AxyP4 and AxyP5 in simulated intestinal fluid within 3 h, respectively. The hepatic metabolic stability of the four modified peptides within 30 min in rat liver S9 preparation was also improved significantly with no metabolism of AxyP5 and threefold (AxyP2 and AxyP4) and eightfold (AxyP3) less metabolism compared with AxyP1 (39 % metabolism). Unlike hydrolysis as the major metabolism of peptides of natural α-amino acids, oxidation mediated by the cytochrome P450 enzymes, especially CYP3A subfamily, to form the corresponding mono-hydroxyl metabolites was the predominant hepatic metabolism of the five α-aminoxy peptides tested. The present findings demonstrate that structural modification can significantly improve the GI and metabolic stability of α-aminoxy peptides and thus increase their potential for therapeutic use in the treatment of chloride channel related diseases.

Assessment of thyroid hormone activity of halogenated bisphenol A using a yeast two-hybrid assay

The thyroid hormone agonist/antagonist activities of halogenated derivatives of bisphenol A (BPA) were assessed using a yeast two-hybrid assay incorporating the human thyroid hormone α (TR α), both with and without possible metabolic activation by rat liver S9 preparation. In the absence of the rat liver S9 preparation, 3,3′,5,5′-tetrabromobisphenol A (TBBPA), 3,3′,5,5′-tetrachlorobisphenol A (TCBPA), and 3,3′,5-trichlorobisphenol A (3,3′,5-triClBPA) exhibited agonist activity, whereas 3-chlorobisphenol A (3-ClBPA), 3,5-dichlorobisphenol A (3,5-diClBPA), 3,3′-dichlorobisphenol A (3,3′-diClBPA), and BPA did not. The activities of TBBPA and TCBPA increased markedly (7.6-fold and 3.1-fold, respectively) after their metabolic activation with the rat liver S9 preparation. TBBPA, TCBPA, and 3,3′,5-triClBPA inhibited the binding of triiodothyronine (T3) to TR α at 2 × 10 −5 M without rat liver S9 treatment and 4 × 10 −6 M with rat liver S9 treatment, demonstrating their T3 antagonist activity. These results revealed that metabolic activation by rat liver S9 significantly increased the agonist/antagonist potential of some halogenated BPAs.

Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls

A series of novel synthetic monohydroxy polychlorinated biphenyls (OH-PCBs) (5 trichloro-, 5 tetrachloro- and 5 pentachloro-compounds) have been characterized (1H and 13C NMR and high resolution MS) and their estrogenic and thyroid hormone activities assessed using a yeast two-hybrid assay, both with and without possible metabolic activation by rat liver S9 preparation. Moderate estrogenic activity was found for 2,3,4′-trichlorobiphenyl-4-ol (compound 5) but this was eliminated when exposed to the S9 mix. 2,2′,3′,4,6-Pentachlorobiphenyl-3-ol (13) and 2′,3,3′,6-tetrachlorobiphenyl-4-ol (10) both showed weak estrogenicity in the absence of the S9 mix. The estrogenicity of compound (10) was enhanced 10-fold by exposure to S9 metabolic activation but that of compound (13) remained unchanged. 2′,4,5′,6-Tetrachlorobiphenyl-2-ol (6) showed strong thyroid hormonal activity (5% of that of T4) whereas 3′,4,6-trichlorobiphenyl-3-ol (4), compound (10) and 2,3′,4,5′,6-pentachlorobiphenyl-3-ol (14) showed moderate activity, and 2′,3,3′,5-tetrachlorobiphenyl-2-ol (8) and 3,3′,5,5′,6-pentachlorobiphenyl-2-ol (11) showed weak activity. The activity of (4) was eliminated by S9 metabolic activation whereas those of (6) and (14) were weakened and that of (10) remained unchanged.

IN VITRO INHIBITION OF CARCINOGEN-INDUCED MUTAGENICITY BY CASSIA OCCIDENTALIS AND EMBLICA OFFICINALIS

Aqueous extracts of Cassia occidentalis Linn. (Leguminoceae) and Emblica officinalis Gaertn. (Euphorbiaceae) were screened for effectiveness in inhibiting mutagenicity of aflatoxin B1 (AFB1) and benzo[a]-pyrene (B[a]P) in the Ames test. Antimutagenicity was evaluated using Salmonella typhimurium strains TA 98 and TA 100. In the assay, metabolic activation of AFB1 (0.5 μg/plate) and B[a]P (1 μg/plate) was mediated by rat liver S9 preparation. Although both plants inhibited mutagenicity, E. officinalis had more inhibitory effect than C. occidentalis. Their action is possibly mediated through interactions with micro somal activating enzymes. Their inhibitory action on chromosomal aberrations together with present results suggest that these plants have potent antimutagenic and anticarcinogenic activities against mutagens requiring metabolic activation.

Identification of in vitro metabolites of indinavir by “intelligent automated LC-MS/MS” (INTAMS) utilizing triple quadrupole tandem mass spectrometry

In an effort to improve the efficiency of the TSQ 7000 LC-MS/MS system for identification of drug metabolites in biological matrices in support of drug discovery programs, a combination of instrument control language procedures for the Finnigan MAT TSQ 7000 mass spectrometer, referred to as INTAMS, were composed. INTAMS was designed to conduct unattended, automatic liquid chromatography/mass spectrometry (LC-MS) and LC-MS/MS analyses of drugs and metabolites in commonly encountered in vitro biological matrices. A novel peak detection algorithm was developed to automatically detect and record the pseudomolecular ions and retention times of chromatographic components, even if not fully resolved. This algorithm was used in combination with an automated technique for predicting the molecular weights of metabolites based on incremental changes of the molecular weight of the parent drug resulting from well-known biotransformation processes. When applied to a sample of an incubation mixture of the HIV protease inhibitor Indinavir with a rat liver S9 preparation, the results obtained by the automatic metabolite detection procedures for LC-MS and LC-MS/MS analyses in real time were the same as those which were determined manually, by a knowledgeable operator.

In vitro metabolism of bladder carcinogenic nitrosamines by rat liver and urothelial cells

In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell α-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybuty)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine α-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N 2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37°C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the α-hydroxylation pathway. Using hepatic S9 fractions, 15N 2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 ± 0.02 nmol/h per mg protein (mean ± S.E.) to 5.78 ± 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N 2 produced by urothelial cell homogenates was about 40–50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the α-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N 2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N 2 production was about 40–50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N 2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N 2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N 2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.

1-Hydroxypyrene as an indicator of the mutagenicity of coal tar after activation with human liver preparations

Liver S9 fractions were prepared from male Wistar rats, either non-induced or induced with Aroclor 1254 and from 5 human kidney transplant donors. The preparations were compared for their ability to metabolize the premutagens present in coal tar to mutagenic metabolites in the Salmonella mutagenicity assay towards strain TA98. Low levels of mutagenicity of coal tar were seen with human S9 preparations. The differences between the S9 mix of the 5 donors in capacity to activate premutagens were approximately 6-fold. The activation of coal tar by rat liver S9 preparations was higher than by the human S9 preparations. The metabolic conversion of pyrene in coal tar to 1-hydroxypyrene by the same human S9 preparations was determined in a parallel assay. 3 human preparations showed a high correlation between the formation of 1-hydroxypyrene and bioactivation of coal tar to mutagenic metabolites. The slope values of the individual regression lines were equal, suggesting that 1-hydroxypyrene is a good indicator for the activation of premutagens present in coal tar.

Metabolism of benz[ j]aceanthrylene (cholanthrylene) and benz[ I]aceanthrylene by induced rat liver S9

The metabolites of benz[ j]aceanthrylene (B[ j]A) produced by incubation with liver S9 proteins from rats induced with Aroclor-1254 and phenobarbital have been identified as: trans-B[ j]A-1,2-dihydrodiol, B[ j]A-9,10-dihydrodiol, B[ j]A-11,12-dihydrodiol, and 10-hydroxy-B[ j]A. The major metabolite formed (58–60%) by both induced S9 preparations was trans-B[ j]A-1,2-dihydrodiol, the cyclopenta-ring dihydrodiol while oxidation at the k-region or the proximal-bay region was minor. There were no statistical differences in individual or total B[ j]A metabolite rates between the 2 induced S9 preparations. B[ l]A was metabolized by Aroclor-1254 and phenobarbital induced rat liver S9 preparations to trans-B[ l]A-1,2-dihydrodiol, B[ l]A-7,8-dihydrodiol, and B[ l]A-4,5-dihydrodiol. The major B[ l]A metabolite formed (28–40%) by both induced S9 preparations was B[ l]A-7,8-dihydrodiol, the k-region dihydrodiol. Cyclopentaring oxidation to trans-B[ l]A-1,2-dihydrodiol was approximately 50% of that observed for k-region oxidation. Both induced S9s produced similar rates of B[ l]A metabolites except for B[ l]A-7,8-dihydrodiol formation which was higher for Aroclor-1254-induced S9.

Mutagenicity of rat-liver S9 to L5178Y mouse lymphoma cells

Rat-liver S9 preparations became highly mutagenic to cultured L5178Y mouse lymphoma cells when the exposure period was increased to 18–24 h or when S9 mix was preincubated in Fischer's medium at 37°C for 19 h and then used to treat the cells for 4 h. Five different S9 preparations (from untreated and Aroclor 1254-treated Fischer 344 or Sprague-Dawley male rats) behaved similarly. S9 mix, which contained 1 mM NADP and 5 mM isocitrate as cofactors, was more mutagenic than S9 alone. Heat treatment of S9 did not destroy its mutagenic activity, but the addition of cofactors no longer stimulated an increase in mutagenicity, as observed with native S9. Treatment with cofactors was not mutagenic. These results implied the involvement of both energy-independent and NADPH-dependent enzymatic changes in S9 mix in producing mutagenic substances. The mutagenic treatments with S9 or S9 mix induced predominantly small TFT-resistant mutant colonies, which suggested that these treatments should be clastogenic to cultured mammalian cells. A warning was given that test chemicals evaluated as mutagenic only in the presence of S9 mix may instead be accelerating the decomposition of S9 mix into mutagens, and it may become necessary to experimentally distinguish between these two mechanisms before a chemical can be regarded as mutagenic.

Metabolic activation of 3-nitroperylene in the Salmonella/S9 assay.

The mutagenicity of 3-nitroperylene (nitroPer), a nitrated polycyclic aromatic hydrocarbon (PAH) has been extensively studied using the Ames Salmonella test. In accord with previous work, nitroPer proved to be a potent frameshift mutagen which requires activation by mixed function oxidases (MFOs) in the microsomes of rat liver S9 preparations. The concentration of S9 required for optimal activation of this mutagen is several times lower than that recommended for routine screening by the Ames test. Studies with the MFO inducers Aroclor 1254, phenobarbital, 3-methylcholanthrene and beta-naphthoflavone, as well as with some MFO inhibitors indicate that both P450b and P450c appear to be involved in the activation of nitroPer. Two non-mutagenic PAHs (perylene and benzo[e]pyrene) inhibited the mutagenicity of nitroPer in a competitive fashion. The mutagenic activity of nitroPer was greatly decreased in a strain of bacteria (TA98/1,8-DNP6) that lacks an acetyltransferase needed for the activation of many nitroarenes. Incubation of nitroPer with microsomes from Aroclor-treated rats plus appropriate cofactors led to the formation of several metabolites which could be separated from one another and from nitroPer by h.p.l.c. Three of these were direct-acting mutagens with Salmonella typhimurium strain TA98, while another required microsomal activation. We postulate that the metabolic activation of nitroPer requires three steps: (i) metabolism by MOF enzymes to yield a ring-oxidized compound which is absorbed by the bacteria; (ii) reduction of this compound to the hydroxylamine by a bacterial nitroreductase; and (iii) O-acetylation of the hydroxylamine to yield a reactive ultimate mutagen.

In vivo and in vitro metabolism of the new anticancer drug bisantrene

The metabolism of bisantrene, a new anthracene anticancer agent active in the treatment of disseminated breast cancer, was studied in vitro using rat liver S9 preparations and in vivo in patients receiving the drug as treatment for their cancers. 14C-ring labeled bisantrene (248 mCi/40 mg) plus cold bisantrene were administered IV to cancer patients (260-340 mg/m2). Fractional urine samples were collected at various time intervals up to 120 h after drug administration and analyzed by HPLC. The percent of total 14C excreted as unchanged parent drug per ml urine ranged from 37 to 79% in the 0 to 24 h samples. The remainder of the radioactivity appeared chromatographically just prior to the bisantrene peak, indicating that compounds more polar than the parent were present as transformation products. Metabolism of bisantrene was also studied in vitro under oxic (O2) and hypoxic (N2) conditions, using commercially available Aroclor 1254 induced rat liver S9 preparations. Following N2 incubation at 37 degrees C for 1 h there was no evidence of metabolism, whereas there was more than 50% decrease in parent drug within 1 h following O2 incubation in the presence of NADPH generating system, suggesting that the metabolic process involves an oxidative reduction. HPLC chromatogram profiles of the mixtures exposed to the activated S9 system indicated that there were at least 3 polar metabolites. In vitro human tumor clonogenic assay showed that the biological activity of bisantrene decreased greater than 4-fold when the drug was incubated with S9 preparations in the presence of NADPH and O2, indicating that the transformation process leads to relatively inactive bisantrene metabolites.

Rat liver S9 preparations contain comutagenic activity

Rat liver S9 preparations contain material which causes enhancement of UV mutagenesis in Escherichia coli WP2. This comutagenic activity is present in S9 preparations from both uninduced and Aroclor-induced rats. Strains of E. coli which are defective in the uvr-dependent excision repair pathway fail to show comutagenic action by S9. The comutagenic material is heat-labile and non-dialyzable, suggesting that it might be protein. This differs from the small amount of mutagenic material present in rat liver S9, as the latter is dialyzable and can be demonstrated in the repair-deficient strain E. coli WP2 s ( uvrA).

Absence of mutagenic activity of fodder proteins in the Ames Salmonella/microsome test

The mutagenicities of fodder proteins (Pekilo, l-lysine and Orsan) were tested towards Salmonella typhimurium in the plate-incorporation assay in the presence or absence of metabolic activation with a rat-liver S9 preparation. Filtrates and 2-, 5- and 10-fold-concentrated filtrates of saline- or ethanol-soluble fodder proteins were tested. No mutagenic activity was observed.

Induction of mitotic gene conversion by browning reaction products and its modulation by naturally occurring agents

Mitotic gene conversion in the D7 strain of Saccharomyces cerevisiae was significantly enhanced by exposure to non-enzymatic browning reaction products. These products were formed during the heating of sugar (caramelization reaction) or sugar-amino acid mixtures (Maillard reaction) at temperatures normally used during the cooking of food. Several modulating factors of this convertogenic activity were identified. These factors included two main groups: (1) trace metals which are widely distributed in the environment; and (2) several cellular enzymatic systems. The convertogenic activities of a heated glucose-lysine mixture and a commercial caramel powder were completely suppressed when yeast were concurrently exposed to these products and to either FeIII or CuII. Equimolar concentrations of MnII or sodium selenite had no effect on the convertogenic activity of the products of either model system. Horse-radish peroxidase, beef liver catalase and rat liver S9 preparations each decreased the frequency of gene conversion induced by the caramel powder and the heated glucose-lysine products. This modulating activity of the enzymes was lost if they were heat-inactivated. These studies indicate the presence of a variety of protective mechanisms which can modify genotoxic components in complex food mixtures.

The activation of mutagens in diesel particle extract with rat liver S9 enzymes.

Reductive activation of nitroaromatic compounds by bacterial enzymes contributes to the direct-acting mutagenicity of diesel particle extracts in the Salmonella mutation assay. In this study, the ability of mammalian enzymes to activate diesel particle extract was investigated with a rat liver S9 preparation using the nitroreductase-deficient strain TA98NR to detect active metabolic products. The use of diesel particle extract at concentrations greater than 50 micrograms per plate inhibited S9 enzyme activations in the mutation assay. In addition, the S9 preparation without NADPH present decreased the residual direct-acting mutagenicity of the particle extract. Activation by S9 enzymes of mutagens in diesel particle extract was evident as a difference in activity between assays with and without NADPH. 1-Nitropyrene, a nitroarene identified in diesel particle extracts, was also activated by NADPH-dependent S9 enzymes. S9 activation of both the particle extract and 1-nitropyrene was detected only when using much lower S9 concentrations than conventionally applied in the Salmonella/S9 assay. S9 enzyme activation of 1-nitropyrene is not merely a consequence of forming a mutagenic amine since 1-aminopyrene was less mutagenic than 1-nitropyrene in these assays. The NADPH-dependent activation of 1-nitropyrene is located in the microsomal fraction of the S9 preparation. However, activation of the diesel particle extract was more evident with the cytosol than with the microsomal fraction, demonstrating the involvement of yet other enzyme systems and extract components.