The effects of di-(2-ethylhexyl)adipate (DEHA) have been compared in female F344 rats and female B6C3F1 mice fed diets containing 0–4.0% DEHA and 0–2.5% DEHA, respectively, for periods of 1, 4 and 13 ...weeks. In both the rat and mouse treatment with DEHA at all time points produced a dose-dependent increase in relative liver weight and hepatic peroxisome proliferation as demonstrated by the induction of peroxisomal (cyanide-insensitive palmitoyl–CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities. The magnitude of induction of peroxisome proliferation was similar in both species. Replicative DNA synthesis was studied by implanting osmotic pumps containing 5-bromo-2′-deoxyuridine during study weeks 0–1, 3–4 and 12–13. After 1 week DEHA treatment hepatocyte labelling index values were increased in rats given 2.5 and 4.0% DEHA and mice given 0.6–2.5% DEHA. While DEHA treatment for 4 and 13 weeks did not increase labelling index values in the rat, a sustained stimulation of replicative DNA synthesis was observed in mice given 1.2 and 2.5% DEHA. The results of this study demonstrate a species difference in the hepatic effects of DEHA, in that at some dose levels DEHA can produce a sustained stimulation of replicative DNA synthesis in mouse but not in rat liver. Sustained cell replication provides a better correlation with the observed formation of liver tumours in chronic studies with DEHA in female mice, but not in female rats, than the magnitude of stimulation of hepatic peroxisome proliferation.
In an effort to determine the total one-year cost of liver transplantation, the underlying drivers of that cost, and any cost differences between alternative immunosuppressive regimens, an analysis ...was performed comparing the average one-year posttransplant charges of 322 patients participating in the "U.S. Multi-center Prospective Randomized Trial Comparing FK-506 to Cyclosporine in Liver Transplantation." Total one-year inpatient charges including all readmissions were examined. Professional fees and outpatient charges were excluded. Costs for tacrolimus drug and blood assays were assumed to be equal to those in the CsA group. For patients completing the study, the tacrolimus group had an average length of stay and average one-year cost seven days (P = .06) and $19,290 (P = .05) lower than the CsA group. The difference in rejection profiles between the two arms seems to largely account for the lower costs. The tacrolimus arm consistently had fewer patients in the more severe rejection groups. Increased incidence and severity of rejection were directly related to higher average lengths of stay and costs of transplantation (P < .001). Tacrolimus immunosuppression during the first year after liver transplantation is more cost-effective than CsA in achieving similar patient and graft survival rates. Differing incidence and severity of rejection can dramatically affect the first-year cost of liver transplantation.
1. The aim was to investigate the effects of some cytochrome P450 (CYP) enzyme inducers on CYP1A and CYP2B subfamily forms in cultured precision-cut rat lung slices.
2. Precision-cut lung slices were ...prepared from male Sprague-Dawley rats and cultured for 24 and/or 48 h in medium containing 0-20 µgml-1 Aroclor 1254 (ARO), 0-50 µM β-naphthoflavone (BNF) and 0-50 µM benzo(a)pyrene (BP).
3. Treatment with ARO, BNF and BP produced significant increases in lung slice whole homogenate 7-ethoxyresorufin 0-deethylase activity.
4. Levels of CYP1A1 apoprotein were markedly increased in lung slice microsomes after treatment for 48 h with either 10 µgml-1 ARO or 5 µM BNF. In contrast, neither ARO nor BNF had any marked effect on levels of CYP2B1/2 apoprotein in 48-h cultured rat lung slice microsomes.
5. Real-time quantitative reverse transcription-polymerase chain reaction methodology (TaqMan®) was used to quantify lung slice CYPlAl and CYP2B1/2 mRNA levels. Rat lung slice CYP1A1 mRNA levels were increased up to 8.3-fold after treatment
for 24h with 2 and 10 µgml-1 ARO, 0.5 and 5 µM BNF, and 20 µM BP. In contrast, treatment with 10 µgml-1 ARO produced only a small 1.6-fold increase in CYP2B1/2 mRNA levels.
6. Precision-cut lung slices are a useful model in vitro system for the assessment of the effects of chemicals on pulmonary CYP forms.
1. The effect of rifampicin on cytochrome P450 isoforms in the CYP1A and CYP3A subfamilies has been studied in 72-h cultured precision-cut human liver slices. 2. In cultured human liver slices 50 ...microM rifampicin induced testosterone 6 beta-hydroxylase activity, but had no effect on 7-ethoxyresorufin O-deethylase and 7-methoxyresorufin O-demethylase activities. 3. Western immunoblotting of liver slice microsomes was performed with antibodies to rat CYP1A2 and human CYP3A4. Compared with control (dimethyl sulphoxide only treated) liver slice microsomes, rifampicin increased levels of CYP3A4 but had no effect on CYP1A2. 4. These results demonstrate that rifampicin induces CYP3A isoforms, but not CYP1A2, in cultured human liver slices. Some variability in the magnitude of induction by rifampicin was observed in the six human liver samples examined. 5. These results demonstrate that cultured human liver slices may be used to evaluate the effects of xenobiotics on CYP3A isoforms.
High doses of Pyrethrins produce liver tumors in female rats. To elucidate the mode of action for tumor formation, the hepatic effects of Pyrethrins have been investigated. Male Sprague-Dawley CD ...rats were fed diets containing 0 (control) and 8000 ppm Pyrethrins and female rats' diets containing 0, 100, 3000 and 8000 ppm Pyrethrins for periods of 7, 14 and 42 days and 42 days followed by 42 days of reversal. As a positive control, rats were also fed diets containing 1200–1558 ppm sodium Phenobarbital (NaPB) for 7 and 14 days. The treatment of male rats with 8000 ppm Pyrethrins, female rats with 3000 and 8000 ppm Pyrethrins and both sexes with NaPB resulted in increased liver weights, which were associated with hepatocyte hypertrophy. Hepatocyte replicative DNA synthesis was also increased by treatment with Pyrethrins and NaPB. The treatment of male and female rats with Pyrethrins and NaPB produced significant increases in hepatic microsomal cytochrome P450 (CYP) content and a marked induction of CYP2B-dependent 7-pentoxyresorufin
O-depentylase and testosterone 16β-hydroxylase activities. Significant increases were also observed in CYP3A-dependent testosterone 6β-hydroxylase activity. The hepatic effects of Pyrethrins were dose-dependent in female rats with 100 ppm being a no effect level and on cessation of treatment were reversible in both sexes. This study demonstrates that Pyrethrins are mitogenic CYP2B form inducers in rat liver. The mode of action for Pyrethrins-induced rat liver tumor formation appears to be similar to that of NaPB and some other non-genotoxic CYP2B inducers of hepatic xenobiotic metabolism.
Agaritine {(β-
N-γ-
l(+)glutamyl-4-hydroxymethylphenylhydrazine)} is present in the common cultivated mushroom
Agaricus bisporus and several agaritine derivatives have been shown to produce tumours ...in experimental animals. In this investigation the metabolism of ring-U-
14Cagaritine has been studied in precision-cut rat, mouse and human liver slices and in precision-cut rat and mouse lung slices. To confirm the functional viability of the tissue slice preparations, the metabolism of 7-ethoxycoumarin was also studied. Liver and lung slices from all species metabolized 50 μM 7-ethoxycoumarin to 7-hydroxycoumarin, which was conjugated with D-glucuronic acid and sulfate. Incubation of rat, mouse and human liver slices, and rat and mouse lung slices with 25 μM
14Cagaritine resulted in a time-dependent formation of metabolite(s), which bound covalently to tissue slice proteins. Agaritine metabolite covalent binding was greater in mouse liver than in rat and human liver slices and was greater in mouse lung than in rat lung slices. No correlation was observed between agaritine metabolite covalent binding and tissue slice γ-glutamyltransferase activity. Additional studies with mouse liver slices showed that
14Cagaritine was also metabolized to a number of unknown polar metabolites. These results demonstrate that agaritine can be metabolized by enzymes present in mammalian liver and lung.
1. Using the recently published crystal structure of a bacterial P450, namely 102 (also termed P450bm3), as a template molecular models of mammalian 2A1, 2A4, 2A5 and 2A6 were constructed. 2. ...Substrate interaction studies demonstrated that in keeping with known catalytic activities the putative binding sites of mouse hepatic P4502A4 and 2A5 oriented testosterone for 15 alpha-hydroxylation and coumarin for 7-hydroxylation respectively. 3. Substrate interaction studies with the putative binding site of human liver P4502A6 demonstrated that coumarin was oriented for 7-hydroxylation. However, in keeping with previous site-directed mutagenesis studies with P4502A4 and 2A5, changing a single phenylalanine residue to leucine in 2A6 gave rise to a mutant enzyme, which could bind testosterone as a substrate for 15 alpha-hydroxylation rather than coumarin. 4. Substrate interaction studies with the putative binding site of rat hepatic P4502A1 suggested that this isoenzyme would hydroxylate coumarin at the 3- rather than at the 7-position. 5. The results of these molecular modelling studies demonstrate that apparently minor modifications to P4502A subfamily amino acid sequences can result in major alterations in enzyme specificity. 6. Molecular modelling is thus a useful technique that can aid in elucidating substrate specificities of P450 isoenzymes and species differences in xenobiotic metabolism. The technique can also be utilized to complement site-directed mutagenesis studies in order to identify critical structural features of P450s and other enzymes.
Eight structurally diverse hypolipidaemic agents have been examined for their ability to induce the microsomal cytochrome P-452-dependent fatty acid hydroxylase system and the enzymes of peroxisomal ...beta-oxidation in rat liver. Using a specific ELISA method, we have shown that the cytochrome P-452 isoenzyme is induced up to ten fold by hypolipidaemic challenge, concomitant with a pronounced elevation of the peroxisomal beta-oxidation enzymes, mirrored by an increase in peroxisomal volume as determined morphometrically. In addition, the induction of cytochrome P-452 is accompanied by a decrease in the activities of cytochromes P-450b and P-450c as measured by benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities respectively, the latter being more extensively reduced by hypolipidaemic treatment. A hypothesis is presented whereby an early biological response is the hypolipidaemic induction of microsomal cytochrome P-452 resulting in omega-hydroxy fatty acids and their subsequent further oxidation to dicarboxylic acids, the latter providing the proximal stimulus for peroxisomal proliferation.
In this study the effect of coumarin on unscheduled DNA synthesis (UDS) in precision-cut human liver slices has been examined. Liver slices from tissue samples from four donors were cultured for 24
...hr in medium containing
3Hthymidine and 0–5.0
m
m coumarin using a dynamic organ culture system and processed for autoradiographic evaluation of UDS. As positive controls liver slices were also cultured with three known genotoxic agents, namely 0.02 and 0.05
m
m 2-acetylaminofluorene (2-AAF), 0.002 and 0.02
m
m aflatoxin B
1 (AFB
1) and 0.005 and 0.05
m
m 2-amino-1-methyl-6-phenylimidazo 4,5-
bpyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with more than five net grains. Compared with control liver slice cultures, treatment with 0.05–5.0
m
m coumarin had no effect on UDS. In contrast, treatment with 0.02 and 0.05
m
m 2-AAF, 0.002 and 0.02
m
m AFB
1 and 0.005 and 0.05
m
m PhIP produced significant increases in the net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05
m
m PhIP. Treatment with 2-AAF, AFB
1 and PhIP also produced significant increases in the number of centrilobular hepatocyte nuclei with more than five net grains. At the concentrations examined neither coumarin, 2-AAF, AFB
1 nor PhIP had any significant effect on replicative DNA synthesis in 24
hr cultured human liver slices. These results demonstrate that coumarin does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice preparations used in this study. The results of this study suggest that coumarin is not a genotoxic agent in human liver.
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