Comparative metabolism of a dieldrin analogue: hepatic microsomal systems as models for metabolism in the whole animal.
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Animals↗
Biomedical subjects
J K Chipman
Publications and source records attributed to J K Chipman.
Microsomal systems as models for in vivo metabolism.
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Animals↗
A technique for chronic intermittent bile collection from the rat.
A technique is described for inserting into the bile duct, a re-entrant cannula with a sampling arm to allow intermittent collections of bile from unanaesthetised, minimally restrained rats, for a period of 30 days or more. The flow rate of bile (5.38+/-0-25 cm3/h/kg) was higher than that previously reported when using simple or re-entrant cannulas. This higher flow rate may be a result of allowing a minimum of 10 days post-operative recovery period before starting collection of bile and of avoiding anaesthesia and other stresses at the time of collection.
Animals↗
Reduction of 2-acetylaminofluorene-induced unscheduled DNA synthesis in human and rat hepatocytes by butylated hydroxytoluene.
Butylated hydroxytoluene (BHT) protected against DNA damage induced in rat hepatocytes by 2-acetylaminofluorene (2AAF) or N-hydroxy 2AAF as shown by a marked reduction of unscheduled DNA synthesis. BHT also inhibited 2AAF-induced DNA damage (as shown by reduced repair) in human hepatocytes. In addition, rats pre-treated with BHT in the diet (0.5% w/w for 10 days) provided hepatocytes which exhibited less unscheduled DNA synthesis than did hepatocytes from control rats when these cells were exposed to either 2AAF or N-hydroxy 2AAF. The results indicate both direct (in vitro) and indirect (by pre-treatment in vivo) inhibitory effects of BHT on the genotoxicity of 2AAF in liver cells, in accord with the reported anti-tumorigenicity in the liver. This effect contracts with a BHT-mediated increase in the efflux of 2AAF-derived mutagens from liver cells which may contribute to enhanced extrahepatic carcinogenesis.
2-Acetylaminofluorene↗
Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay).
Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Port Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P < 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).
Animals↗
The cytochrome P450 1A gene (CYP1A) from European flounder (Platichthys flesus), analysis of regulatory regions and development of a dual luciferase reporter gene system.
Concensus primers designed to CYP1A-conserved regions were used to amplify a 1.3 kb probe from flounder genomic DNA via polymerase chain reaction (PCR). A 14-kb clone was isolated from a flounder genomic library constructed in lambda FIXII. Of this clone, 8 kb was sequenced, including 3 kb of upstream sequence. The predicted amino acid sequence showed closest similarity to plaice CYP1A1 (98%). Gene structure conformed to the seven exons and six introns common to previous CYP1A sequences, but intron lengths were not conserved. Concensus sequences corresponding to xenobiotic and other response elements as well as TATA, CAAT and GC boxes were identified. Upstream sequence (3.5 kb) including the first exon and intron up to the putative start codon were amplified via PCR and inserted upstream of the luciferase gene in a pGL3 reporter gene construct. The HepG2 mammalian hepatoma cell line was transiently co-transfected with the flounder CYP1A reporter gene construct and the pRL-CMV internal control construct. The maximal induction upon exposure to 100 nM 3-MC was 4.4-fold in comparison with carrier-treated cells. Use of deletion constructs resulted in loss of inducibility.
Animals↗
Analysis of mutations in the p53 tumor suppressor gene and Ki- and Ha-ras proto-oncogenes in hepatic tumors of European flounder (Platichthys flesus).
DNA from five tumors, three other-injured livers and two normal liver tissue samples from the European flounder were analyzed for mutations in exons 5-8 of the tumor suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. No tumor-specific mutations were identified by direct sequencing and single-strand conformation polymorphism of these genes. A number of silent polymorphisms were noted in p53. In addition to a need for more extensive analyses of flounder liver tumor samples for ras or p53 mutations, other cancer-related genes should be investigated.
Animals↗
Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons.
Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.
Adaptation, Physiological↗
Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site.
Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (sigma7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYPIA-immunopositive protein were 1.5 fold higher (P < 0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P < 0.05) and higher levels of DNA damage (1.2 fold; P < 0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P < 0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.
Animals↗
Metabolism of 3-phenoxybenzoic acid and the enterohepatorenal disposition of its metabolites in the rat.
The absorption, metabolism and disposition of 3-phenoxybenzoic acid (3PBA) and its 4'-hydroxylated derivative (4'HO3PBA) have been investigated in intact and bile duct-cannulated rats. Both acids are rapidly and extensively eliminated in the urine as the O-sulfate ester of 4'HO3PBA (4'HOSO2O3PBA). Biliary excretion was 45% of the dose when 3PBA was administered at 100 mg/kg, compared with 16% at 10 mg/kg. The major biliary metabolites, identified by mass spectrometry, were 3PBA glucuronide and the ether and ester glucuronic acid conjugates of 4'HO3PBA. These biliary metabolites were of minor importance in the urine. At a dose of 100 mg/kg, an apparent excretion balance figure approximately 120%, as determined from the extent of elimination of radioactivity in the urine (72%) of intact animals and bile of cannulated rats (45%) (only 7% appeared in the feces), suggests that the biliary glucuronides decompose and/or are enzymically cleaved in the gastrointestinal tract to the respective benzoic acids. The latter are subsequently reabsorbed and undergo further metabolism, principally to the sulfate ester which is excreted in the urine. This enterohepatorenal disposition of 3PBA has been confirmed by suppression of the gut microflora with antibiotics in vivo, which dramatically reduces the extent of deconjugation of biliary glucuronides and hence reabsorption of the free benzoic acids. The deconjugation by gut bacteria has also been demonstrated in vitro.
Animals↗