Safety and psychological impact of sailing adventure therapy among Veterans with substance use disorders.

OBJECTIVES: Many Veterans suffer from substance use disorders (SUDs). Treatment challenges include poor treatment engagement and high relapse rates. Complementary interventions have the potential to enhance both. This study was a preliminary evaluation of sailing adventure therapy (SAT) for this population. DESIGN: Retrospective chart review. Participants in the intervention were 22 Veterans (20 male, 2 female) aged 22-65 who entered a Veterans Administration residential SUD treatment program. All subjects had two or more SUDs, and many had psychiatric (95%) and/or medical (77%) comorbidities. The age, gender and diagnosis-matched control group (n = 22) received residential SUD treatment as usual (TAU) in the same program but without SAT. SETTING: Residential SUD treatment program at a Veterans Administration Medical Center. INTERVENTION: Sailing adventure therapy. MAIN OUTCOME MEASURES: Positive and Negative Affect Schedule (PANAS), State Trait Anxiety Inventory six-item short form (STAI: Y-6 item), Acceptance and Action Questionnaire II (AAQ II), Five Facet Mindfulness Questionnaire (FFMQ) and a locally developed patient survey. Outcome comparison among SAT plus TAU group versus TAU - only group included measures of successful completion of residential SUD treatment program as well as psychiatric hospitalizations and/or residential SUD treatment program readmissions within 12 months. RESULTS: Neither physical injuries nor increases in anxiety or negative affect occurred, as measured by the PANAS (positive change, p = 0.351; negative change, p = 0.605) and the STAI: Y-6 item (p = 0.144) respectively. There was no significant change in FFMQ (p = 0.580) but a significant increase occurred in AAQ II scores (p = 0.036) indicating an increase in psychological flexibility. Survey responses indicated the participants perceived the experience to be both pleasurable and calming. The preliminary outcome evaluation revealed a significant between-group difference (X2 = 5.34, DF = 1, p = 0.02, r = 0.35) indicating participating in SAT was associated with a greater likelihood of successfully completing residential SUD treatment. However, there were no significant between-group differences in number of psychiatric hospitalizations (X2 = 1.09, DF = 1, p = 0.29, r = 0.16) or residential substance abuse treatment program readmissions (X2 = 0.23, DF = 1, p = 0.64, r = 0.07) in the 12 months after discharge from the program. CONCLUSIONS: Preliminary evidence suggests that SAT is physically safe and not associated with increased anxiety or negative affect. Participant's perceptions of the experience were positive. Preliminary outcome measures suggest associations between participation in SAT and increased psychological flexibility as well as successful completion of a residential SUD treatment program. Further research is indicated to determine whether SAT may be developed as an effective complementary intervention for Veterans with SUDs.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

Drug stimulated biotransformation of hormonal steroid contraceptives: clinical implications.

On the basis of well documented biochemical and pharmacological data about the influence of drug mediated enzyme induction on the biotransformation of natural and synthetic sex steroids, practical consequences for hormonal steroid contraception are described and discussed. Clinical reports dealing with this problem are still sparse. The clinical symptoms of drug stimulated biotransformation of hormonal steroid contraceptives are characteristic. Spotting or breakthrough bleeding are observed and in the extreme case conception may occur despite the regular intake of the contraceptive. The appearance of these symptoms differs from one individual to another. With a strong enzyme inducer, bleeding disorders can be provoked artifically in 50 to 60% of the women receiving hormonal contraceptive treatment. The range of drugs which stimulate biotransformation of hormonal contraceptives with consequent loss of their biological effectiveness is not completely known. For practical purposes, it is recommended that bleeding disturbances under hormonal steroid contraception in a previously regular cycle be regarded as loss of reliability; they should be remedied and taken as a sign to search for uncontrolled drug taking.

Influence of 3-methylcholanthrene on liver nucleolar and nucleoplasmic activities of protein kinases and RNA polymerases.

The experiments were designed to investigate some details of the action of 3-methylcholanthrene (3-MC) on the regulation of transcription. After a single intraperitoneal dose of 3-MC a significant increase in the activities of both nucleolar and nucleoplasmic protein kinases in hepatic cells of young rats was found. The maximal stimulation took place 24 hr after the administration of 3-MC and the extent of activation was much greater in the nucleolar fraction. There is a significant elevation of the activities of both functional forms, free and template-engaged, of RNA polymerase A 24 hr after a single injection of 3-MC. Free and engaged forms of extranucleolar RNA polymerase B show a different behaviour: after 24 hr of 3-MC administration the engaged form is markedly enhanced while the activity of the free enzyme shows a significant decrease. The more moderate increase in total RNA polymerase B activity is obviously preceded by a transfer of the enzyme from 'free' to 'engaged' form. Since the enhancement of protein kinase activities was accompanied by the stimulation of nuclear RNA polymerases we suggest that both kinds of enzymes are involved in an epigenetic mechanism of the inducing action of 3-MC on cytochrome P1-450.

Developmental aspects of xenobiotic transformation.

In most laboratory animals monooxygenases are apparently absent or barely detectable in fetal organs until just before birth. In this contribution hepatic cytochrome P-450-dependent reactions in the rat are considered only. The results are interpreted on basis of the reaction scheme of Estabrook. To avoid methodological pitfalls the basic kinetics for all reactions investigated have been investigated with liver preparations from newborn and adult rats. The low monooxygenase activity of rat liver during the perinatal period can be observed even under optimal conditions for the in vitro enzyme assay. There are different developmental patterns for various reactions O-demethylation of codeine, phenazone-hydroxylation, first and second steps on N-demethylation of amidopyrine, N-demethylation of ethylmorphine. There are marked differences not only in Vmax but also in the postnatal development of Km and the inductibility by phenobarbital. Thus the existence of a different cytochrome P-450 is evident also by this approach. The low monooxygenase activity of rat liver during the perinatal period is not due to a lack of NADPH or NADH, to an age-dependent NADPH cytochrome P-450 reductase activity or to an age-dependent NADH-cytochrome P-450 reduction. Moreover this low activity is not due to an insufficient mitochondria-endoplasmic reticulum interaction. It is accompanied by low delta Amax after addition of a typical type I substrate (hexobarbital) and by a small amount of metyrapone-binding centers: it can be explained by a smaller percentage of active cytochrome P-450 in comparison to adult rat liver.

Chlorinated drinking water is mutagenic and causes 3-methylcholanthrene type induction of hepatic monooxygenase.

Acid/neutral fractions of 4 chlorinated drinking water samples were tested for mutagenicity in the Ames' test and injected intraperitoneally to 10- and 20-day-old Wistar rats at doses of 200 and 100 liters of water/kg body weight. Cytochrome P-450 mediated enzyme activities of ethylmorphine-N-demethylase (EMND), 7-ethoxycoumarin-O-deethylase (ECOD), 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PEROD) were determined in the 9000 g supernatant fraction of liver homogenate. EROD was introduced by the concentrates. The induction was related to the mutagenic activity. About 4-fold increase in activity was observed with the most mutagenic sample. PEROD was also slightly enhanced. EMND and ECOD activities were not affected by the lower dose, but the higher dose caused inhibition of 30-40%. Although the extracts were not toxic to bacteria, they were unexpectedly toxic to rats. It is concluded that the samples contained 3-methylcholanthrene (3-MC) type inducer(s).

Hepatic actions of levonorgestrel: correlations between biochemical and morphological findings.

The influence of the synthetic sexual steroid levonorgestrel (LN) on rat liver in various doses and at different structural levels was investigated. A slight reactive hepatosis was found by histological examination after administration of LN in a dose of 10 mg per kg body wt. The same dose caused exclusively distinct lesions of the mitochondria, however, only in centrilobular parenchymal cells, whereas in the periportal hepatocytes only the lipid droplet content appears somewhat elevated. LN decreased the total glutathione content of the liver. The mitochondrial glutathione was decreased more intensively. One mg/kg body wt. of LN decreased the cytochrome P-450 content, but 10 mg/kg body wt. increased ethyl-morphine N-demethylation and 7-ethoxycoumarin O-deethylation activities. Distinct correlations could be shown between the biochemical changes and the ultrastructural findings.

Estradiol, testosterone, dehydroepiandrosterone and androstenedione: novel derivatives and enantiomers. Interactions with rat liver microsomal cytochrome P450 and antioxidant/radical scavenger activities in vitro.

Interactions of 27 steroids, among them 17 derivatives such as ethers, sulfates and amidosulfonates derived from 17 beta- and 17 alpha-estradiol, from testosterone and alpha- and beta-dihydrotesosterone and from dehydroepiandrosterone with rat liver microsomal cytochromes P450 (P450) were investigated in vitro by assessing binding to P450 and effects on P450 mediated monooxygenase functions as measured by different model reactions: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD) and ethylmorphine N-demethylation (EMND). With the exception of 17 alpha-estradiol-3-dimethylamidosulfonate, estrone, its -3-methylether and -3-amidosulfonate and testosterone, all other steroids displayed type I or reverse type I binding to P450. All steroids inhibited EROD activity in micromolar concentrations. An additional strong inhibition of ECOD and EMND activities was only demonstrated for the androgens and progestins. Estriol, estrone and mestranol displayed less inhibitory actions on the model reactions than estradiol. No major differences in comparison to the parent compounds were noted with the other derivatives. The only exceptions were 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol, which displayed stronger effects than estradiol, and dehydroepiandrosterone-3-sulfate, which was less effective than dehydroepiandrosterone. Possible antioxidant properties of the steroids were examined by the stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) using rat liver microsomes. Additionally, the influence on rat whole blood chemiluminescence (WB-CL) was assessed. All the estrogens, but not their methylethers and amidosulfonates inhibited LPO in micromolar concentrations. The effects on the other oxidase model reactions or on WB-CL were less distinct. Only ethinylestradiol and 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol displayed a strong inhibitory action on all model reactions. With the exception of dehydroepiandrosterone-3-sulfate, which in general had only weak effects, the androgen and progestin derivatives, in contrast, strongly decreased H2O2 formation and LM- and LC-CL, but were mostly ineffective on LPO and WB-CL.

Influence of skin and muscle lesions on the hepatic cytochrome P450 function in 10 and 60 day-old male Wistar rats.

In 10 and 60 day-old male Wistar rats skin lesions of different extents and muscle lesions lead to decreases in cytochrome P450 concentrations and monooxygenase activities (ethylmorphine N-demethylation and ethoxycoumarin O-deethylation) at least up to 7 days after operation. The extent of the depression was related to the extent of the lesion and independent of the nature of the tissue involved.

The influence of systemic hypoxia and reoxygenation on the glutathione redox system of brain, liver, lung and plasma in newborn rats.

The concentrations of reduced (GSH) and oxidized glutathione (glutathione disulfide, GSSG) in lung, liver, brain and plasma of newborn rats were investigated under the condition of reversible hypoxia. Brain and lung of newborn rats seem to be susceptible to reversible hypoxia. We found an increase in GSSG concentration after hypoxia in these organs. This alteration of the GSH-GSSG redox system was reversible within 2 hours of reoxygenation. A second increase in cerebral GSSG concentration after 4 hours of reoxygenation was connected with fasting during the experiment. In the liver we found a hypoxia dependent decrease in the GSH level, followed by a decrease in GSSG concentration. The increased GSSG concentrations in lung and brain are accompanied by an enhancement of plasma GSSG concentration.

Luminol and lucigenin amplified chemiluminescence and lipidperoxidation with brain microsomes from rats during ontogenetic development.

The chemiluminescence (CL) amplifiers luminol (LM) and lucigenin (LC) react with different reactive oxygen species (ROS) in dependence on the ROS generating system used. With liver microsomes LMCL indicates predominantly superoxide anion radicals, whereas LCCL is mainly a measure for hydroxyl radical formation or of reactive organic radicals. With brain microsomes only LCCL, but not LMCL could be measured. For both brain microsomes from newborn (both sexes) and 60 day-old (male) rats LCCL is dependent on protein and NADPH concentration, activity in newborns being only 15% compared with young adult rats. As compared with liver microsomes 10-fold higher protein concentrations are needed to obtain comparable LCCL, whereas the NADPH demand is the same as with liver. A distinct ontogenetic development was demonstrated: low activities in the fetus, in newborn and 10-day-old rat are followed by higher activities with increasing age, after a maximum at an age of 60 days a decline was observed. Microsomal lipidperoxidation (LPO) was measured as formation of thiobarbituric acid reactive substances (TBARS) and was also dependent on protein and NADPH concentration. Unexpectedly, LPO with brain microsomes from newborn rats did not show any developmental variation.

Nerval influences on liver cytochrome P450.

In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation.

Peroxidative status and glutathione content of the brain in normal weight and intra-uterine growth-retarded newborn piglets.

The peroxidative and glutathione status as well as the production of reactive oxygen species were studied in the brain of normal weight (NW) and intra-uterine growth-retarded (IUGR) newborn piglets. In NW as well as IUGR newborn piglets reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxides, iron stimulated lipid peroxidation, H2O2 production and lucigenin and luminol amplified chemiluminescence are very similar in the different brain regions, with one exception. In the cerebellum, higher GSH concentration, higher superoxide anion generation, lower levels of lipid peroxides and a tendency toward a lower capacity of H2O2 production were seen. But the intra-uterine growth retardation to body weights of half the average body weights of the respective litter did not influence the peroxidative status and the GSH/GSSG equilibrium in the brain of newborn piglets.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 mediated monooxygenase functions and on oxidative state.

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on P450 mediated monooxygenase functions in liver and spleen 9,000 g supernatants were assessed by measuring the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD), and ethylmorphine N-demethylation (EMND). Additionally, the influence on the oxidative state was investigated by assessing the liver and spleen tissue content of lipid peroxidation (LPO) products and of reduced and oxidized glutathione (GSH;GSSG). The livers of both solvent treated transplant recipients and control rats displayed regular EROD, ECOD, PROD and EMND activities. After AAL treatment EROD and EMND activities within the livers were not affected, but ECOD and PROD activities were increased. BBZ administration caused a decrease in EROD and EMND activities, ECOD activity remained unaffected, and PROD activity was even increased. CCl4 and TAA administration caused a strong reduction in the activity of all four model reactions. Spleens of control rats displayed almost no P450 mediated monooxygenase functions, independent whether the rats had been treated with the cytotoxins or not. In the transplant containing spleens, however, significant EROD and ECOD, but hardly any PROD or EMND activities were seen. After AAL administration EROD activity was not affected in the transplant containing spleens, but ECOD activity was increased. BBZ treatment led to a decrease in EROD and an elevation in ECOD activity. CCl4 and TAA strongly reduced the activity of both of these model reactions. The tissue content of LPO products within livers and transplant containing spleens was significantly increased after BBZ and CCl4 treatment. An elevation in LPO products was also seen in the spleens of the control rats due to CCl4 administration. Tissue GSH and GSSG content in both livers and transplant containing spleens were strongly reduced after BBZ treatment. After CCl4 administration only a significant decrease in liver GSSG contents was seen. TAA treatment caused a reduction in the GSH and GSSG content in the spleens of both transplant recipients and control rats, but not in the livers. From these results it can be concluded, that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA on P450 dependent monooxygenase functions and on oxidative state are exerted in the ectopic intrasplenic liver cell transplants in a similar way as in normal orthotopic liver.

Glutathione homeostasis and turnover in the totally hepatectomized rat: evidence for a high glutathione export capacity of extrahepatic tissues.

Glutathione (GSH) homeostasis and turnover were investigated in totally hepatectomized (HX) rats. A technique is described to remove the liver totally, with preservation of the hepatic portal and vena caval vasculature. Euglycemia could be maintained with hourly infusions of 50 mg 100 g-1 b.m. of glucose after bolus i.v. injection of glucose at the same dose. The efficiency of the animal model was demonstrated by examination of paraclinical blood parameters: progressive increases in total plasma bilirubin and alkaline phosphatase activity were noted after HX; the other parameters tested were predominantly in the normal range during the observation period of 6 hours. Histological examination revealed an acute but reversible impairment of intestine and kidneys. These results indicate that the surgical procedure and postoperative care were able to secure sufficient physiological conditions for the experiments over a longer period. 3 to 6 hours after HX we observed a decreased but stable plasma GSH level in anhepatic rats (about 50% of the control value). The GSH levels of brain and kidney were not changed. With increasing time period after HX the heart and lung GSH levels were depressed. A small depression of muscle GSH concentration was observed 4 and 6 hours after HX. A progressive increase in the concentration of oxidized glutathione was seen in brain and kidney. Our observations could be indicative for a high GSH export capacity of extrahepatic tissues contributing about 50% of the total GSH influx into circulation. Probably, the skeletal musculature is an important GSH origin for plasma.

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes.

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content.

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 in the periportal areas was even decreased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.

Age dependent different influence of carbon tetrachloride on biotransformation of xenobiotics, glutathione content, lipid peroxidation and histopathology of rat liver.

15- and 60-day-old male rats were treated with different doses of CCl4 orally. 24 h later cytochrome P-450 (P450) concentration, 7-ethoxyresorufin O-deethylation (EROD) and 7-pentoxy-resorufin O-deethylation (PEROD) activities were determined. Whereas P450 and EROD are lowered to the same extent in both ages, PEROD shows a more pronounced inhibition in the livers of younger rats. The formation of endogenous lipid peroxides (measured as thiobarbituric acid reactive substances) is drastically increased only in the livers of young rats. The hepatic glutathione (GSH) content was unaffected by CCl4 treatment whereas oxidized glutathione is more increased in the livers of adult rats. This can be caused by a higher activity of GSH-peroxidase in the livers of adult rats. The changes in NADPH-induced lipid peroxidation and chemiluminescence correlate partially with the changes in P450 and biotransformation reactions. Histopathologically the liver damage is more extensive in suckling rats. The necrosis is localized predominantly in the perivenous tissue, which has normally the highest activities of toxification and detoxification enzymes.

Influence of the xanthine derivative denbufylline and the anti-inflammatory agent nabumetone on microsomal free radical production and lipid peroxidation in rat liver.

The influence of denbufylline, nabumetone and its main metabolite BRL 10,720 on iron stimulated lipid peroxidation (LPO), cytochrome P 450 dependent H2O2 and chemiluminescence (CL) production was investigated in rat liver microsomes in vitro (10(-5)-10(-3) M) and in vivo after treatment of rats (5-300 mg/kg b.m. orally on three consecutive days). In rat liver slices the release of thiobarbituric acid reactants (TBAR) was measured after 1 hour of incubation with the drugs. Denbufylline, nabumetone and BRL 10,720 exerted a significant inhibition of iron stimulated LPO in vitro. Nabumetone showed the strongest antioxidative activity, which was also seen in liver slices. These antioxidative effects were not found after in vivo treatment of rats. Denbufylline (10(-3) M) additionally inhibited H2O2 formation and the luminol and lucigenin amplified CL in vitro. Unexpectedly, nabumetone increased H2O2 formation both in vitro and in vivo, but in vitro only lucigenin amplified CL. BRL 10,720 increased microsomal H2O2 production in vivo. Moreover, BRL 10,720 enhanced CL in vitro and in vivo significantly, which is interpreted as an increase of the production of superoxide anion radicals and other reactive oxygen species such as H2O2, but lipid peroxidation in liver microsomes was not enhanced. These results suggest that denbufylline, nabumetone and BRL 10,720 in contrast to the in vitro effects did not exert antioxidative activities after treatment of rats. On the contrary, BRL 10,720 was found to support the formation of reactive oxygen species in liver microsomes.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: inducibility of cytochrome P450 dependent monooxygenase functions by beta-naphthoflavone, phenobarbital and dexamethasone.

In the present study the effects of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on cytochrome P450 (P450) dependent monooxygenase functions were investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of 60-90 days old adult male syngenic Fisher 344 inbred rats. 2, 4 or 6 months after surgery, transplant recipients and age matched controls were orally treated with BNF (1x50 mg/kg body weight (b.wt.)), PB (1x50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. P450 mediated monooxygenase functions were measured in spleen and liver 9000 g supernatants by three model reactions for different P450 subtypes: ethoxyresorufin O-deethylation (EROD; 1A), ethoxycoumarin O-deethylation (ECOD; 1A, 2A, 2B), and ethylmorphine N-demethylation (END; 3A). Spleen weights were significantly higher in transplanted rats, compared to controls, at all three time points after surgery. Induction with PB or DEX, and in some cases also with BNF, lead to a significant increase in liver weights of transplant recipients and control rats independent of the time after transplantation. In contrast, there was no influence on spleen weights due to BNF or PB. At all time points after surgery, with DEX a marked decrease in body weights, weights of adrenal glands and of lymphatic organs like thymus glands and spleens was observed, with the weights of the transplant containing spleens being still higher in comparison to control organs. Spleens of control animals displayed nearly no P450 mediated monooxygenase functions neither without nor with induction. After transplantation, however, significant EROD and ECOD, but hardly any END activities were seen in the host organs at all three time points after surgery. In transplant containing spleens EROD and ECOD were significantly increased after BNF or PB treatment at all three time points after surgery, and ECOD after DEX administration, but at 4 and 6 months after transplantation only. END was only induced after DEX treatment at 6 months after transplantation. With the livers of both transplant recipients and control rats EROD and ECOD were increased after BNF induction and EROD, ECOD, and END after PB treatment at all three time points after transplantation. After DEX administration END was significantly enhanced only at 2 and 4 months after transplantation, ECOD was decreased at 2 and 4 months, and EROD was diminished at all three time points after surgery. Transplantation of fetal liver tissue suspensions into the spleens did not influence monooxygenase functions and their inducibility within the respective livers of the animals. These results demonstrate that transplanted liver cells originating from syngenic fetal liver tissue suspensions display P450 dependent monooxygenase functions which are, simi lar to normal adult liver, inducible by BNF, PB and DEX. Both monooxygenase functions and their inducibility within the transplant containing spleens display quantitative and qualitative developmental changes.