Induction of unscheduled DNA synthesis in primary rat hepatocytes by benzidine-congener-derived azo dyes in the in vitro and in vivo/in vitro assays

The genotoxicity of the benzidine-congener-derived azo dyes. Direct Blue 1 ( DB1 ), Direct Blue 14 ( DB14 ), Direct Brown 95 ( DB95 ), and Direct Red 46 ( DR46 ) was studied in the in vitro and in vivo/in vitro unscheduled DNA synthesis (UDS) assays in primary rat hepatocytes to determine if in vivo metabolism of these compounds was required for induction of UDS. Hepatocytes were isolated, cultured, and treated with the azo dyes and [3H]thymidine (in vitro assay); alternatively, in the in vivo/in vitro assay, rats were intubated with the azo dyes, the hepatocytes isolated at 17 h after dosing and incubated in a medium containing [3H]thymidine. UDS was quantified by an autoradiographic method. None of the azo dyes induced UDS in the in vitro assay. However, DR46 did induce marginal, but significant UDS in 1 experiment (1.2 net grains at 500 micrograms/ml media). No significant UDS was observed when DR46 was tested in a subsequent in vitro assay. In the in vivo/in vitro assay, DB95 (100 mg/kg), DB14 (125 mg/kg), and DR46 (100 mg/kg) induced significant UDS (12, 2.1, and 3.5 net grains, respectively). None of the azo dyes tested was mutagenic in the Salmonella/microsome assay in the presence and absence of rat liver enzymes. Therefore, in vivo reduction of azo dyes, presumably by the gut microflora, is a requirement for the genotoxicity of these azo dyes in the primary rat hepatocyte UDS assay.     

An assessment of the in vivo rat hepatocyte DNA-repair assay

The in vivo rat hepatocyte autoradiographic assay for unscheduled DNA synthesis (UDS) described by Mirsalis et al, and its in vitro counterpart described earlier by Williams have been employed by us for 4 years. Our experience is that the in vivo assay performs as described in the literature. We have therefore concentrated in this initial paper on the key practical factors we have found to govern the assay sensitivity and reproducibility. This has been achieved by a discussion of the assay performance with two potent rat hepatocarcinogens [the novel azo compound 6-dimethylaminophenylazobenzthiazole (6BT) and the reference agent 2-acetylaminofluorene (2AAF)] and a non-carcinogen of similar structure to 6BT [5-dimethylaminophenylazoindazole (51)]. Assay responses were compared with the effect of these chemicals in the Salmonella mutation assay. We conclude that the in vivo liver UDS assay has a critical role to play as a complement to rodent bone marrow cytogenic assays when conducting assessment studies on agents defined as genotoxic in vitro. However, the in vivo assay is resource-consuming and false results could consequently arise due to incomplete evaluations. Methods to counteract this danger are discussed and criteria for assessing weak UDS responses are suggested.     

Relative sensitivity of 32P-postlabelling of DNA and the autoradiographic UDS assay in the liver of rats exposed to 2-acetylaminofluorene (2AAF)

Groups of male Alderley Park rats were dosed concomitantly with 2-acetylaminofluorene (2AAF) by gavage at doses between 0.01 mg/kg and 40 mg/kg, and livers sampled 2-72 h later. The liver of one group of animals was perfused to yield hepatocytes which were assayed in vitro for unscheduled DNA synthesis (UDS) via incorporation of tritiated thymidine and autoradiography. DNA was extracted from the livers of the other group and DNA adduct levels determined using the 32P-postlabelling technique. The major C-8 2-aminofluorene/guanosine adduct and 3 minor adducts were quantitated, enabling the relative sensitivity of the 2 techniques to be compared. A dose- and time-related UDS response was observed, which, at the most sensitive time-point (12 h) enabled DNA repair to be discerned at a dose level of 0.1-1 mg/kg of 2AAF, a response classified as formally positive at 5 mg/kg 2AAF. Only the C-8 adduct, as determined by 32P-postlabelling, was discernible at 0.01 mg/kg of 2AAF, although other adducts were visible on autoradiograms at higher dose levels. It is concluded that as part of a well-defined dose response, UDS can be discerned with confidence for doses of 2AAF between approximately 0.1 and 5 mg/kg, and DNA adducts for doses of 2AAF between approximately 0.01 and 1 mg/kg. Discernible UDS for 2AAF in the rat liver is apparent at approximately 13 DNA (total) adducts/10(8) nucleotides, or approximately 8 DNA (C-8) adducts/10(8) nucleotides. The presumed C-8 2-acetylaminofluorene/guanosine adduct, prepared by reaction of 2-acetoxy-2-acetylaminofluorene (2AAAF) with DNA, was a significant but unreliable marker of 2AAF/DNA adducts in the rat liver in vivo. DNA repair did not appear to remove DNA adducts selectively, and adducts remained in DNA when discernible DNA repair had ceased.     

Metabolic activity in primary cultures of fish hepatocytes

In aquatic toxicology, isolated liver cells from fish can be used as a tool to generate initial information on the hepatic metabolism of xenobiotics, and on the mechanisms of xenobiotic activation or deactivation. This isolation of teleost liver cells is achieved by enzymic dissociation, and monolayer cultures of fish hepatocytes in serum-free medium maintain good viability for 3-8 days. During in vitro culture, fish liver cells express stable levels of phase I and phase II enzymes, such as cytochrome P4501A or glutathione S-transferase, and the cells show an induction of biotransformation enzymes after exposure to xenobiotics. The xenobiotic metabolite pattern produced by fish hepatocytes in vitro is generally similar to that observed in vivo. Limitations to more-intensive application of cultured fish hepatocytes as a screen in aquatic hazard assessment are partly due to the rather limited scope of existing studies, i.e. the focus on one particular species (rainbow trout), and on one particular biotransformation enzyme (cytochrome P4501A), as well as a lack of comparative in vitro/in vivo studies.     

Investigation of the stereoselective in vitro biotransformation of glutethimide by high-performance liquid chromatography and capillary electrophoresis

Due to our interest in drugs with a glutarimide structure, we reinvestigated the stereoselectivity of the in vitro biotransformation of the chiral hypnotic-sedative drug glutethimide. Glutethimide enantiomers were separated on a preparative scale by HPLC on cellulose tris(4-methylbenzoate) as chiral stationary phase. The enantiometric purity was higher than 99%. A reversed-phase HPLC method was developed to determine the metabolites of glutethimide. After incubations with rat liver microsomes both enantiomers formed 5-hydroxyglutethimide as the main metabolite, as well as additional metabolites, of which some were formed stereoselectivity. Mass spectrometry of the unknown metabolites indicated a hydroxylation in the ethyl side chain for two of the metabolites. A third metabolite was tentatively identified as desethylgutethimide.     

Synthesis and degradation of eicosanoids in primary rat hepatocyte cultures

Arachidonic acid metabolites may play an important role in liver physiology, yet hepatocyte prostaglandin synthesis has not been characterized extensively. We used RIA to study production and clearance of several eicosanoids in confluent primary cultures of rat hepatocytes in serum-free, hormonally-defined medium. Under basal, unstimulated conditions 6-keto-PGF1 alpha (spontaneous breakdown product of prostacyclin) and 13,14-dihydro-15-keto-PGE (DHK-PGE, a metabolite of PGE) accumulated in the culture medium. Hepatocytes cleared 6-keto-PGF1 alpha, thromboxane B2, and DHK-PGE from the medium. Production of eicosanoids by primary cultures appeared resistant to indomethacin and several other cyclooxygenase inhibitors. This apparent resistance to indomethacin was not caused by rapid metabolism of indomethacin, by failure of the drug to enter hepatocytes, or by insensitivity of hepatocyte cyclooxygenase to the drug. Metabolism of PGE to DHK-PGE may be saturated under in vitro conditions. Hepatocytes can synthesize significant amounts of eicosanoids, although they are probably less active in this regard than are non-parenchymal cells.     

Investigation of the stereoselective in vitro metabolism of the chiral antiasthmatic/antiallergic drug flezelastine by high-performance liquid chromatography and capillary zone electrophoresis

An achiral HPLC method using a silica gel column as well as two independent chiral analytical methods by HPLC and capillary zone electrophoresis (CZE) were developed in order to investigate the in vitro metabolism of the racemic antiasthmatic/antiallergic drug flezelastine. The chiral HPLC analysis was performed on a Chiralpak AD column, which also allowed the simultaneous separation of the N-dephenethyl metabolite. The chiral separation by CZE was achieved by the addition of β-cyclodextrin to the run buffer. The stereoselectivity of the in vitro biotransformation of flezelastine was investigated using liver homogenates of different species. Depending on the species, diverse stereoselective aspects were demonstrated. The determination of the enantiomeric ratios of flezelastine and of N-dephenethylflezelastine after incubations of racemic flezelastine with liver microsomes revealed that porcine liver microsomes showed the greatest enantioselectivity of the biotransformation. (−)-Flezelastine was preferentially metabolized. After incubations with bovine liver microsomes the enantiomer of N-dephenethylflezelastine formed from (+)-flezelastine dominated. Incubations of the pure enantiomers of flezelastine with induced rat liver microsomes resulted in the stereoselective formation of a hitherto unknown metabolite, which was only detected in samples of (+)-flezelastine. Initial structure elucidation of the compound indicated that the new     

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.     

Species differences in cytotoxic and genotoxic effects of phenacetin and paracetamol in primary monolayer cultures of hepatocytes

The cytotoxic and genotoxic effects of phenacetin and paracetamol were examined in monolayer cultures of hepatocytes isolated from the mouse, hamster, rat and guinea pig. No marked increase in unscheduled DNA synthesis (UDS) after exposing hepatocytes from any of the species to phenacetin was observed. At cytotoxic concentrations of paracetamol, an increased UDS in mouse hepatocytes in vitro was observed. Pretreatment of the mice by inducers of drug-metabolizing enzymes, such as 3-methylcholanthrene and Aroclor 1254, lowered the concentration threshold for the toxic responses. With rat hepatocytes only a minor increase in UDS was noted, while with hepatocytes from hamsters and guinea pigs in fact a decrease was seen. The narrow range observed between the cytotoxic and genotoxic effects of paracetamol makes it difficult to predict whether the initial DNA damage could lead to a mutation or whether the cells will die before the mutation is expressed. With respect to the cytotoxic effects, hamster hepatocytes were found to be most susceptible to paracetamol, followed by mouse, while rat and guinea pig were less affected. These data were in accordance with in vivo findings (Davis et al., 1974), indicating the potential value of hepatocyte culture when screening for possible liver toxic substances.     

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.     

Effect of serum lipoproteins on stereoselective halofantrine metabolism by rat hepatocytes

Experimental hyperlipidemia has shown to decrease cytochrome P450 3A4 and 2C11 expression and to increase liver concentrations and the plasma protein binding of halofantrine (HF) enantiomers. The present study examined the effect of hyperlipidemic (HL) serum on the metabolism of HF enantiomers by primary rat hepatocytes. Hepatocytes from normolipidemic (NL) and HL (poloxamer 407 treated) rats were incubated with rac-HF in cell media with or without additional rat serum (5%). In those incubations with rat serum, the hepatocytes were preincubated or coincubated with serum from NL or HL rats. Rat serum-free hepatocyte incubations served as controls. Stereospecific assays were used to measure HF and desbutylhalofantrine (its major metabolite) enantiomer concentrations in whole well contents (cells + media). Concentrations of desbutylhalofantrine were not measurable. The disappearance (apparent metabolism) of (-)-HF exceeded that of antipode, but HF metabolism did not differ between hepatocytes from NL and HL rats. Coincubation of HL rat serum with NL hepatocytes caused a significant decrease in the disappearance of (-)-HF, whereas in HL hepatocytes, a substantially decreased apparent metabolism was noted for both enantiomers. Compared with NL serum, (-)-HF disappearance was significantly lowered upon preincubation of NL hepatocytes with HL serum. A combination of factors including diminished drug metabolizing or lipoprotein receptor expression, and increased plasma protein binding in the wells, may have contributed to a decrease in apparent metabolism of the HF enantiomers in the presence of lipoproteins from HL rat serum.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes.

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Diethyldithiocarbamate inhibits scheduled and unscheduled DNA synthesis of rat thymocytes in vitro and in vivo--dose-effect relationships and mechanisms of action

In vitro as well as in animal models, diethyldithiocarbamate (DDC) modifies the tumoricidal activity of some antineoplastic agents. To gain further information about the mechanism of action of DDC, we measured (i) in vitro and (ii) in vivo changes in DNA synthesis of rat thymocytes. (i) In vitro, the scheduled (SDS) and unscheduled (UDS) incorporation of [3H]thymidine ([3H]dT) into DNA of rat thymic cells were biphasically inhibited in a dose range of 1-1000 micrograms DDC/ml. The UV-induced UDS was totally suppressed by 10 and 100 micrograms DDC/ml. (ii) In vivo, 1-4 h following intraperitoneal administration of 250-1000 mg DDC per kg body wt., SDS and UDS were inhibited up to about 80% in a dose-dependent manner. Nucleoid sedimentation, uptake of [3H]dT into the cells, and the pattern of phosphorylation of the intracellular [3H]dT following DDC treatment did not reveal any differences to the controls. A possible effect of DDC treatment on the ribonucleotide reductase and the DNA polymerase alpha is suggested.     

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro

To determine if hormone-induced events leading to ovulation an granulosa cell luteinization might be associated with changes in the surface configuration of granulosa cells we have studied the morphology of granulosa cells from the preovulatory follicles both in vivo and in vitro. In vivo, granulosa cells in follicles from rats primed with estradiol and FSH developed bulbous protrusions termed blebs in response to injected hCG. The blebs were restricted to the adluminal granulosa cells which possess the least number of receptors for hCG. When granulosa cells from follicles of rats primed with estradiol and FSH were cultured in vitro, in the absence of serum, approximately 10% of the cells formed blebs. In the presence of 10% rat or fetal calf serum, nearly 90% of the cells formed blebs by 18 hr. Serum-induced bleb formation was prevented by 1 mM dibutyryl cycle-AMP plus 0.5 mM methyl isobutyl xanthine and by cytochalasin B (25 mug/ml), while 0.1 muM colchicine had no effect. Fibronectin at 25 mug/ml increased bleb formation three-fold over control values in serum-free medium. When hCG was included in serum containing medium, the majority of the cells remained smooth without any blebs. Thus, in contrast to its action in vivo, hCG inhibited the formation of blebs in vitro. When the cells incubated in the presence of dbcAMP plus methyl isobutyl xanthine in serum-containing medium, none of the cells formed blebs. One explanation for the seemingly opposite actions of hCG in vivo and in vitro is that hCG might act to alter the permeability of the pre-ovulatory follicles, and thereby allow the admission of serum. The admitted serum component(s) could then induce the formation of blebs on receptor-deficient adluminal cells that did not have elevated cAMP concentrations. The results suggest that fibronectin and/or other serum components, act to induce microfilament-dependent, cAMP-inhibited bleb formation on granulosa cells in vivo and in vitro.     

Influence of the thromboxane-mimetic U-46619 and the prostacyclin metabolite 6-keto prostaglandin E1 on vasoconstriction induced by noradrenaline and 5-HT in rat mesenteric vasculature

The aim of this study was to investigate the effects of U-46619, a thromboxane A2-mimetic, and 6-keto prostaglandin E1 (6-keto PGE1) a biologically active metabolite of prostacyclin, on vasoconstrictor responses to noradrenaline and 5-hydroxytryptamine (5-HT). In vitro, U-46619 (3-100 nmol/l) amplified responses to both noradrenaline and 5-HT in a concentration-dependent manner. This effect was not caused by an increase in the affinity of the alpha-adrenoceptor for noradrenaline because U-46619 (100 nmol/l) did not alter the pA2 of phentolamine. In vivo, U-46619 (100 nmol/l) induced vasoconstriction and consequently significantly shifted the log-concentration-effect curves to noradrenaline and 5-HT upward in an additive manner. 6-Keto PGE1 (1 mumol/l) did not affect either perfusion pressure or vasoconstriction in response to noradrenaline in vivo. The study highlights some differences in responses between in vitro- and in vivo-perfused mesentery.     

Estrogen metabolism in rat liver microsomal and isolated hepatocyte preparations--II. Inhibition studies

The abilities of various inhibitors and metabolism modifiers to alter the metabolism of estradiol and the irreversible binding of estradiol to proteins were examined in subcellular microsomal incubations and in intact hepatocyte preparations. In studies with rat liver microsomal preparations containing estradiol and an NADPH-generating system, the irreversible binding of radiolabeled steroid metabolite(s) to the microsomal proteins was 77.59 pmol/mg/min (SD 6.1; 7.6% of total steroid). 2-Bromoestradiol and 4-bromoestradiol, inhibitors of estrogen 2-hydroxylase, effectively decreased this irreversible binding of radiolabeled estradiol metabolite(s) to microsomal proteins to 17 pmol mg-1 min-1 (2.1% of total estradiol). These haloestrogens were also effective inhibitors in the intact hepatocyte cells, decreasing the amounts of organic metabolites, aqueous-soluble conjugates, and protein-bound materials. The HPLC radiochromatograms of the organic-extracted fractions from the 2 h hepatocyte incubations demonstrate that the catechol estrogen products, i.e. 2-hydroxyestrogens and 2-methoxyestrogens, were present in lower amounts in the incubations containing the bromoestrogens than in control incubations containing no inhibitor. Ascorbic acid and cysteine, general modifiers of oxidative pathways of metabolism, also affected estradiol metabolism in microsomal and hepatocyte preparations. Both these agents were able to decrease the irreversible binding of estradiol to proteins in the microsomal assays. Ascorbic acid decreased the general metabolism of estradiol in the hepatocyte incubations but did not decrease irreversible binding to proteins. The addition of cysteine to the hepatocyte incubation resulted in an increased metabolism of estradiol and the production of more aqueous-soluble radiolabeled metabolites than the control incubations; however, cysteine did not decrease the amounts of estradiol metabolite(s) irreversibly bound to proteins. Investigations of steroid metabolism in the isolated hepatocytes thus provide an effective in vitro technique for examining the overall oxidative, reductive, and conjugative pathways that are functional in the liver and enables one to investigate the abilities of inhibitors, regulators, and modifiers to affect the metabolic processes. Also, these hepatocyte studies demonstrate that the inhibitors of estrogen 2-hydroxylase, 2-bromoestradiol and 4-bromoestradiol, can enter and act in the intact cells. Consequently, these agents may be useful pharmacological probes for examining the functions of catechol estrogens in other tissues.     

Assessement of the relation between the initial viability and the attachment of freshly isolated rat hepatocytes used for the in vivo/in vitro DNA repair assay (UDS)

Crucial steps of the in vivo/in vitro DNA repair assay (UDS) are the hepatocyte isolation procedure and the establishment of the hepatocyte cultures. Since the attachment of the isolated hepatocytes on the surface of the culture vessel is an essential prerequisite for the in vitro part of this assay to yield scorable autoradiograms, we assessed the relation between the initial viabilities of hepatocyte preparations and the resulting attachment efficiencies from 286 rats. The initial viability was determined by means of the trypan blue dye exclusion assay. The actual cell number was corrected for the viability and a constant number of 2.5 × 10⁵ viable cells were seeded into each well of gelatinized six-well dishes. The amount of adherent cells was determined after a 1.5-h attachment period using a recently described modification (Fautz et al., 1991) of the neutral red dye absorption assay. The attachment is described by the optical density at 540 nm obtained after the elution of neutral red from the adherent cells (OD₅₄₀ value).To facilitate a comparison of the data we divided the 286 animals into eight arbitrary viability groups. The mean values of the viability groups were 53.1, 62.2, 66.3, 68.4, 70.9, 73.6, 76.9, and 84.0% living cells. Although there was a great interindividual variation, the resulting mean OD₅₄₀ values were nearly uniform, about 0.5, in all eight groups, regardless of the initial viability of the hepatocytes.UDS data obtained from 46 animals treated with the positive control chemical 2-acetylaminofluorene demonstrated that there was no correlation between the in vitro DNA repair capacity and the initial viability or the attachment efficiency of the hepatocytes.Our results suggest that (i) great interindividual differences exist between the attachment of particular cell preparations with no regard to the initial viability, (ii) the correction of the cell number for viability leads to relatively uniform OD₅₄₀ mean values and (iii) for an in vivo/in vitro UDS assay even cell suspensions with relatively low viabilities can be used since they will yield adherent cultures which are capable of DNA repair synthesis. The latter item often allows a reduction in the number of animals required for this in vivo assay because it is not necessary to perform repeated experiments because of low viability preparations.     

Ethanol-induced inhibition of leukotriene degradation by omega-oxidation

omega-Oxidation of leukotrienes is a major pathway in the degradation and inactivation of these proinflammatory mediators. Ethanol inhibited this process in vivo and in vitro. In rat liver in vivo the catabolism of LTE4 to omega-carboxylated leukotrienes was inhibited by 57% by an ethanol dose of 25 mmol/kg body mass administered intragastrically. The site of inhibition was the oxidation of omega-hydroxy-N-acetyl-LTE4 to omega-carboxy-N-acetyl-LTE4 resulting in an accumulation of omega-hydroxy-N-acetyl-LTE4 and of N-acetyl-LTE4. Analogous results were obtained for the oxidative degradation of LTB4 and omega-hydroxy-LTB4 in rat hepatocyte suspensions. Ethanol, at a concentration of 12.5 mmol/l (0.07%; by vol.), caused 68% inhibition of the oxidation of omega-hydroxy-LTB4 by 50% in hepatocyte suspensions. The conversion of omega-hydroxy-LTB4 to omega-carboxy-LTB4 by rat and human liver cytosol was inhibited by ethanol with half maximal concentrations of 100 mumols/l and 300 mumols/l, respectively. Our measurements indicate that direct interference by ethanol of the omega-oxidation of leukotrienes as well as an increased NADH/NAD+ ratio induced by ethanol led to the inhibition of leukotriene degradation. The impairment of leukotriene inactivation in the liver by ethanol may contribute to the development of the inflammatory reaction in acute alcoholic liver disease.     

Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes

A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells.UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle.Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine.The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.     

Pharmacokinetic,metabolic stability,plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors

N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, synthesized from the avanafil analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC was highly bound to plasma proteins in rats, dogs and human tested and the mean values for PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for 60?min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no significant inhibitory effects on major CYP450 enzymes, and had no significant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, t_max was 6 and 0.5?h, respectively. The clearance for NHPPC was 1.19 and 1.46?L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect.