Immunological Detection and Quantitation of DNA Adducts Formed by 4-Aminoazobenzene Species in vivo

Antibodies to 3-methoxy-4-aminoazobenzene (3-MeO-AAB) and 2-methoxy-4-aminoazobenzene (2-MeO-AAB) DNA adducts were raised in rabbits against in vitro-adducted DNA samples. The enzyme-linked immunosorbent assay (ELISA) was used to determine the sensitivity and specificity of these antibodies. They proved highly specific for the modified DNA used as the immunogen, but cross-reacted with each other. Moreover, they showed cross reactivity with DNA modified by 4-( o -tolylazo)- o -toluidine, but not by other carcinogens, such as 4-aminobiphenyl or 4-nitroquinoline 1-oxide. The 50% inhibition level of antibody binding in the competitive ELISA was at 10–20 fmol of modified base per assay (equivalent to 1–2 adducts per 10⁶ bases). Immunohistochemical staining indicated that these antibodies bind specifically to nuclear components of the liver in rats given either 3-MeO-AAB or 2-MeO-AAB at the dose of 50 mg/kg body weight.     

Preferential inhibitions of hepatic P450IA2 expression and induction by lead nitrate in the rat

Male F344 rats were treated with lead nitrate and changes inthe expression and induction of P450IA subfamily enzymes anda placental form of glutathione-S-transferase (GST-P) in theliver were assessed by means of a bacterial mutation test, immunoblottingwith a monoclonal antibody reactive to P450IA1/IA2 and anti-GST-Psera and Northern blotting with P450IA2 cDNA as a probe. Treatmentof rats with lead nitrate (20, 50 or 100 µmol/kg bodywt) decreased P450IA2 mRNA and protein in the liver in the dose-dependentfashion and also decreased the microsomal activity for P450IA2-dependentmutagenization of aromatic amines. Pretreatment of rats withlead nitrate suppressed the inductions of both P450IA2 mRNAand protein by an inducer of P450IA subfamily enzymes in theliver. In addition, amount of the induced P450IA2 was decreasedalong with increase in that of the induced GST-P.     

A nonspecific colonic ulcer occurring after hepatectomy: Report of a Case

We herein report the case of a 53-year-old man with a nonspecific acute colonic ulcer whose liver function deteriorated after he had undergone hepatectomy. He was referred to our hospital for a hepatoma caused by hepatitis B virus and a right hemihepatectomy was performed. His liver function was poor after the operation, and minor complications such as pleural effusion and biliary fistula developed. A large amount of melena was seen 29 days after the hepatectomy and he developed hemorrhagic shock. Superior mesenteric arteriography revealed pooling of blood in both the hepatic flexure of the ascending colon and the cecum. An emergency right hemicolectomy was performed. There was a 5 x 1-mm ulcer 18 cm distal to the ileocecal valve. Numerous erosions were observed to be scattered throughout the colonic mucosa. The patient recovered slowly and was discharged 6 months after the hepatectomy. This is the first report of an acute colonic ulcer that could have been caused by liver dysfunction.     

Decrease in serum thyroxine level by phenobarbital in rats is not necessarily dependent on increase in hepatic UDP-glucuronosyltransferase.

We have previously reported that there is a poor correlation between increase in the levels of UDP-glucuronosyltransferases, UGT1A1 and UGT1A6, and decrease in the levels of serum total thyroxine (T4) and free T4 in phenobarbital (PB)-treated rats, although the PB-induced decrease in rats is generally thought to occur through induction of the UDP-glucuronosyltransferase (T4-UDP-GT: UGT1A1 and UGT1A6). In the present study, to clarify a relationship between the decrease in serum T4 level and the increase in the T4-UDP-GT activity by PB in rats, we examined the relationship using Gunn rats, a mutant strain of Wistar rats deficient in UGT1A isoforms. Levels of serum total T4, free T4, and total triiodothyronine (T3) were markedly decreased not only in Wistar rats but also in Gunn rats 1 day after the final administration of PB (80 mg/kg i.p., once daily for 4 days), and no significant difference in magnitude of the decrease between Wistar and Gunn rats was observed. On the other hand, the level and activity of T4-UDP-GT were significantly increased by treatment with PB in Wistar rats but not in Gunn rats. Furthermore, significant decrease in the activity of hepatic type I iodothyronine deiodinase, which mediates the deiodination of T4 and T3, by PB treatment was observed in both Wistar and Gunn rats. In addition, no significant change in the level of serum thyroid-stimulating hormone, the activity of hepatic sulfotransferase, and the binding of [125I]T4 to serum transthyretin and albumin by PB treatment was observed in either Wistar or Gunn rats. In conclusion, the present results demonstrate that the decrease in serum total T4 level by PB in Gunn rats is not dependent on the increase in hepatic T4-UDP-GT activity and suggest that even in Wistar rats, the PB-induced decrease in serum T4 level does not occur only through increase in hepatic T4-UDP-GT.     

Blood pressure after treatment with sodium–glucose cotransporter 2 inhibitors influences renal composite outcome: Analysis using propensity score-matched models

Aims/IntroductionSodium–glucose cotransporter 2 inhibitors (SGLT2i) improve renal outcome in patients with type 2 diabetes mellitus, but the mechanism is not fully understood. The aim of this retrospective study was to assess the association of achieved blood pressure with renal outcomes in Japanese type 2 diabetes mellitus patients with chronic kidney disease.Materials and MethodsWe assessed 624 Japanese type 2 diabetes mellitus patients with chronic kidney disease taking SGLT2i for >1 year. The patients were classified as those with post‐treatment mean arterial pressure (MAP) of ≥92 mmHg (n = 344) and those with MAP of <92 mmHg (n = 280) for propensity score matching (1:1 nearest neighbor match with 0.04 of caliper value and no replacement). The end‐point was a composite of progression of albuminuria or a decrease in the estimated glomerular filtration rate by ≥15% per year.ResultsBy propensity score matching, a matched cohort model was constructed, including 201 patients in each group. The incidence of renal composite outcome was significantly lower among patients with MAP of <92 mmHg than among patients with MAP of ≥92 mmHg (n = 11 [6%] vs n = 26 [13%], respectively, P = 0.001). The change in estimated glomerular filtration rate was similar in the two groups; however, the change in the albumin‐to‐creatinine ratio was significantly larger in patients with MAP of <92 mmHg.ConclusionsIn Japanese type 2 diabetes mellitus patients with chronic kidney disease, blood pressure after SGLT2i administration influences the renal composite outcome. Blood pressure management is important, even during treatment with SGLT2i.     

Altered Expression of Hepatic CYP1A Enzymes in Rat Hepatocarcinogenesis

Hyperplastic nodules of the liver were induced by treating male F344 rats with a combination of diethylnitrosamine and partial hepatectomy. The livers were examined for the expression of cytochrome P450 (CYP) enzymes, mainly CYP1A1 and CYP1A2; the amount and activity of the enzymes in the nricrosomes were assessed by enzymatic and immunological methods. Levels of CYP1A mRNAs were assayed by Northern blotting. In the liver bearing hyperplastic nodules, the total amount of microsomal CYP enzymes decreased to about 50% of the control. The microsomal activities for the CYP1A2-mediated activation of carcinogenic heterocyclic amines decreased to about 20% of the corresponding controls, in association with decreases in the levels of mRNA and protein of CYP1A2. Furthermore, the inducibility of CYP1A2 by CYP1A inducers such as 3-methoxy-4-aminoazobenzene and 3-methylcholanthrene was also decreased at the mRNA, protein and activity levels. On the other hand, CYP1A1 enzyme, which was undetectable in control rat liver, appeared in the liver bearing hyperplastic nodules, but its inducibility by a CYP1A inducer decreased slightly. The present findings indicated that individual CYP1A enzymes are differently regulated, and the expression of CYP1A2 is reduced preferentially in the liver bearing hyperplastic nodules.     

Decreased Levels of 2-Amino-3-methylimidazo[4,5-f]quinoline-DNA Adducts in Rats Treated with β-Carotene, α-Tocopherol and Freeze-dried Aloe

To assess mechanisms of chemoprevention of hepatocarcinogenesis by trans -β-carotene (β-C), DL-α-tocopherol (α-T), and freeze-dried whole leaves of Kidachi aloe (Aloe), formation of 2-amino-3-methylimidazo[4,5- f ]quinoline (IQ)-DNA adducts was measured by ³²P-post-labeling analysis, and CYP1A1 and CYP1A2 protein levels were analyzed by ELISA. Group 1 rats were fed diet containing 0.02%β-C, 1.5%α-T or 30% Aloe over an 8-day period, while group 2 was given basal diet alone. On day 7, all animals were subjected to two-thirds partial hepatectomy (PH). Twelve hours after PH, they received a single dose of the carcinogenic food pyrolysate IQ (100 mg/kg) intragastrically, to initiate hepatocarcinogenesis. Rats were killed 6, 12, 24 and 48 h after IQ administration. The levels of adducts, expressed as relative adduct labeling values in rats treated with β-C, α-T and Aloe, were decreased as compared with the control group at hour 24 (36 h after PH), with a significant difference in the case of the β-C group (46.4% of the control value). Similarly, all showed a tendency for decrease at hour 48. Furthermore, the levels of CYP1A2, known to be responsible for activation of IQ, showed a significant reduction at hour 24. It is concluded that β-C, and possibly also α-T and Aloe, have the potential to reduce IQ-DNA adduct formation, presumably as a result of decreased formation of active metabolites. The results may explain, at least in part, the previously observed inhibitory effects of these compounds on induction of preneoplastic hepatocellular lesions.     

Changes in the Quantity and Activity of Cytochrome P-450 Isozymes in Primary Cultured Rat Hepatocytes

Hepatocytes from male Spragne-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro , and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes - test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.