Hepatic UDP-glucuronyltransferase Research Articles

Changes in hepatic metabolic enzyme activities and biliary excretion of 4-nitrophenol in streptozotocin induced diabetic rats

Activity of hepatic metabolic enzymes of glucuronidation and sulfation of 4-nitrophenol (PNP) and biliary excretion of its glucuronide (PNP-G) and sulfate (PNP-S) conjugates have been investigated in control and streptozotocin (STZ)-induced diabetic rats. 500 μM PNP solution was luminally perfused in a cannulated jejunal loop for 90 minutes. It was found that biliary excretion of PNP-G was significantly decreased in the diabetic rats. This effect of STZ could be completely reversed by administration of rapid-acting insulin. Activity of hepatic UDP-glucuronyltransferase and β-glucuronidase was also depressed by the STZ pretreatment. Administration of insulin antagonized the inhibitory action of STZ on UDP-glucuronyltransferase, but the reduced activity of β-glucuronidase was not reversed. Biliary excretion of PNP-S was also depressed in the diabetic rats. Whereas, different effects of insulin administration were observed. Namely, the lower biliary excretion rate of PNP-S was not changed after administration of insulin. Activity of the sulfotransferase and the arylsulfatase enzymes was not altered either by STZ pretreatment or by insulin administration. Biliary excretion of PNP was also significantly depressed by STZ and this depression was not changed after insulin administration. The results call attention to hepatobiliary circulation of low molecular weight xenobiotics and their glucuronide and sulfate conjugates.

UGT1A10 drives tissue‐specific differences in the clearance of the chiral tobacco carcinogen, NNAL

Tobacco‐specific nitrosamines (TSNAs) are among the most potent carcinogens found in tobacco, with 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) the most potent and one of the most abundant. NNK is extensively metabolized to the equally carcinogenic chiral 4‐ (methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanol (NNAL). (S)‐NNAL exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its enantiomeric counterpart, (R)‐NNAL. This differential carcinogenic potential makes it increasingly important to know which UGT enzymes target specific NNAL enantiomers for glucuronidation. NNAL is detoxified primarily by four hepatic UDP‐glucuronyltransferase (UGT) enzymes: UGTs 1A9, 2B7, 2B10 and 2B17; each of these UGTs have been shown to exhibit different stereo‐specificity for the formation of glucuronides of the different NNAL enantiomers. The goal of the present study was to identify the UGTs involved in extra‐hepatic NNAL metabolism and examine differences in the stereospecific glucuronidation of NNAL between tissue types with different UGT expression. Large differences in stereo‐specificity for (S)‐ versus (R)‐NNAL were identified upon comparison of NNAL‐glucuronides (Gluc) formed in reactions of extra‐hepatic UGT1A10‐over‐expressing cell microsomes upon incubation with racemic NNAL, with an enantiomeric preference for (R)‐NNAL as a substrate when compared to (S)‐NNAL. When UGT1A10‐over‐expressing cell microsomes were incubated with pure NNAL enantiomers, large difference in kinetics were observed, with a Km=1.5 mM and Vmax=4.6 pmol/min/mg for (R)‐NNAL, vs a Km>16 mM and Vmax=1.3 pmol/min/mg for (S)‐NNAL. Human liver microsomes preferentially formed (S)‐NNAL‐Gluc while microsomes from human intestine, larynx, esophagus, tonsil, floor of mouth, and tongue all preferentially formed (R)‐NNAL‐Gluc upon incubation with racemic NNAL. Liver and intestine were shown to express UGTs 1A9, 2B7, and 2B17 while intestine, unlike liver, has also been shown to express UGT1A10. All other tissue types analyzed were shown to express only UGTs 1A10 and 2B17, with the exception of esophagus that also expressed UGT2B7. These data indicate that UGT1A10 may drive the difference in differential tissue‐specific clearance of the specific NNAL enantiomers known to induce tobacco‐related cancers.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

울금(Curcuma longa L.)이 고지방·고콜레스테롤 식이 흰쥐에서의 담즙산 및 UDP-glucuronyl transferase 활성에 미치는 영향

The current study examined the effect of turmeric powder on bile acid and UDP-glucuronyl transferase activity in rats fed a high-fat and -cholesterol diet. Sprague-Dawley male rats weighing 120±10 g were randomly assigned to a normal diet group (N group) and a high-fat and -cholesterol diet group (HF group), which was further divided into a high-fat and high-cholesterol with a 2.5% tumeric powder supplement group (TPA group) and 5% turmeric powder-supplemented group (TPB group). Body weight gain and food efficiency ratio were significantly increased in the N group as compared to the HF group, but they were significantly decreased in turmeric-supplemented groups as compared to the HF group. The total serum cholesterol and TG contents of the turmeric-supplemented groups were decreased as compared to those of the HF group. Especially, the TPB group was significantly decreased as compared to the HF group. The serum LDL-cholesterol and AI of the turmeric-supplemented groups were decreased as compared to the HF group. The hepatic triglyceride contents of all groups supplemented with the tumeric powder were significantly decreased as compared to the HF group. The hepatic UDP-glucuronyl transferase activity of the turmeric-supplemented groups was increased as compared to the HF group. In particular, the TPB group was significantly increased as compared to the HF group. The serum total bile acid contents of the turmeric-supplemented groups were increased as compared to the HF group. These results suggest that tumeric has powerful health benefits that are created via UDP-glucuronyl transferase activity, bile acid, and lipid metabolism.

고추씨 에탄올 추출물이 콜레스테롤 흡착능 및 UDP-glucuronyl transferase 활성에 미치는 영향

본 연구에서는 고추씨 에탄올추출물이 콜레스테롤 흡착능 및 UDP-glucuronyl transferase 활성에 미치는 영향을 관찰하였다. In vitro 콜레스테롤 흡착능에서는 대조군에 비해 고추씨 에탄올추출물 모든 농도에서 유의적으로 증가하였다. In vivo에서 실험동물은 체중 <TEX>$100{\pm}10$</TEX> g 내외의 Sprague-Dawley계 수컷 흰쥐를 사용하였으며, 실험군은 정상식이를 공급한 정상군과 고지방 고콜레스테롤 식이 실험군으로 나누고 고지방 고콜레스테롤 실험군을 다시 고추씨 에탄올추출물을 공급하지 않은 군(HF groups), 0.1% 공급한 군(HEA group), 0.2%을 공급한 군(HEB group) 및 0.5%을 공급한 군(HEC group), 5군으로 나누고 각 군당 8마리씩 3주간 사육하였다. 체중증가량은 정상군에 비해 HF 군에서 유의적으로 증가하였고 고추씨 에탄올추출물을 공급한 모든 군에서 HF 군에 감소하였으나 유의적인 차이는 없었다. 식이효율은 고추씨 에탄올추출물을 각각 0.1%, 0.2% 및 0.5% 공급한 군에서는 HF군에 비해 유의적이지는 않았지만 감소하는 경향을 보였다. 간조직의 총콜레스테롤 및 중성지질 함량은 정상군에 비해 HF 군에서 유의적으로 증가하였고 HF 군에 비해 HEA, HEB 및 HEC군에서는 유의적이지는 않았지만 다소 감소하는 경향을 나타내었다. UDP-glucuronyl transferase 활성에서는 정상군에 비해 HF 군에서 증가되었고, 고추씨 에탄올추출물 공급군인 HEC 군에서는 HF 군에 비해 유의적으로 증가되어 가장 높은 활성을 나타내었다. 혈청중의 총콜레스테롤 및 중성지방 농도는 정상군에 비해 HF 군에서 유의적으로 증가하였고, HEA, HEB 및 HEC 군에서 HF 군에 비해 감소하였다. HDL-콜레스테롤은 정상군에 비해 HF 군에서 유의적으로 감소하였으나 HEA, HEB 및 HEC 군에서는 HF 군에 비해 정상군 수준으로 증가하는 경향을 보였다. 반면, LDL-콜레스테롤은 HF 군에서 정상군에 비해 유의적으로 증가하였으나 HEA, HEB 및 HEC 군에서는 모두 유의적 감소를 나타내었다. 대변중의 총콜레스테롤 함량은 정상군에 비해 HF 군에서 유의적으로 증가하였고 HF 군에 비해 특히 고추씨 에탄올추출물 공급군 HEB 군 및 HEC 군에서 증가하는 경향을 나타내었으나 유의적인 차이는 없었다. 중성지질 함량은 정상군에 비해 HF 군에서는 유의적이지는 않았지만 증가되었고, 고추씨 에탄올추출물을 공급한 HEB군 및 HEC 군에서 HF 군에 비해 다소 증가하는 경향을 나타내었다. 따라서 고추씨 에탄올 추출물은 콜레스테롤 흡착능에서 효과적으로 작용하였으며 고지방 고콜레스테롤 식이 흰쥐의 UDP-glucuronyl transferase 활성 및 지질대사에 효과적으로 작용하였음을 규명하였다. The purpose of this study was to investigate the effects of ethanol extracts from red pepper seeds on cholesterol adsorption capacity and UDP-glucuronyl transferase activity. In vitro cholesterol adsorption capacity of 2%, 5% and 10% ethanol extracts from red pepper seed groups were significantly higher than that of the control group. Sprague-Dawley strain male rats weighing <TEX>$100{\pm}10$</TEX> g were randomly assigned to one normal diet N group and experimental groups fed high fat and high cholesterol diet, which were divided into HF (0.0%), HEA (0.1%), HEB (0.2%), and HEC (0.5%) groups according to the amount of ethanol extracts from red pepper seeds added to their basal diet. The body weight gain in the HF group was higher than that in the N group, and those in the HEA, HEB and HEC groups were lower than that in the HF group However, there were no statistically significant differences among the all the groups. The hepatic triglyceride and total cholesterol contents in the N group was significantly lower than that in the HF group, and those in the HEA, HEB and HEC groups were lower than that in the HF group. The hepatic UDP-glucuronyl transferase activity in the N group was lower than that of the HF group and those in the HEA, HEB and HEC groups were lower than that of the HF group. The serum total cholesterol and triglyceride contents of the HF group were significantly higher than that of the N group, and those of the HEA, HEB and HEC groups were lower than that of the HF group. The serum HDL-cholesterol contents in all groups supplemented with the ethanol extracts from red pepper seeds were significantly higher than that of the HF group. The serum LDL-cholesterol contents of the HF group were significantly higher than that of the N group, and those of the HEA, HEB and HEC groups were lower than that of the HF group. The fecal total cholesterol contents were significantly higher in the HF group compared to the N group, and those of the HEB and HEC groups were lower than that of the HF group. The fecal triglyceride contents in the N group was higher than that of the HF group, and those of the HEA, HEB and HEC groups were lower than that of the HF group. This study suggested that the ethanol extracts from red pepper seeds have powerful health benefits by the UDP-glucuronyl transferase activity and lipid metabolism.

무청분말이 콜레스테롤식이를 공급한 흰쥐의 장기능 및 분변 중 중성지질 및 Sterol 배설에 미치는 영향

본 연구에서는 고콜레스테롤 흰쥐에 무청파우더를 공급함으로써 분변을 통한 지질배설과 장기능 개선에 미치는 효과와 그 작용기전을 규명하고자 간조직의 UDP-glucuronyl transferase 활성변화와 분변중의 지질 및 sterol류의 조성을 관찰하였다. 실험동물은 체중 100 g내외의 Sprague-Dawley종 수컷 흰쥐를 이용하였으며, 정상군과 고콜레스테롤 식이 실험군으로 나눈 후 정상군은 다시 무청파우더를 공급하지 않은 군(N group), 무청파우더를 공급한 군(NR group)으로 나누고 고콜레스테롤 식이 실험군은 무청파우더를 공급하지 않은 군(HC group)과 무청파우더를 각각 2.5%(HRL group), 5%(HRM group), 10%(HRH group)씩 공급한 군으로 나누었다. 식이와 식수는 자유섭식 시켰으며, 4주 간 사육한 후 희생시켰다. 분변량은 무청공급군들에서 무청의 공급량이 증가할수록 많은 배변량을 보였다. 수분함량 역시 HC군에 비해 무청공급군들에서 많은 함량을 보였다. 분변중의 총지질, 총콜레스테롤 및 TG 함량은 HC군에 비해 무청공급군들에서 현저하게 증가되었다. 분변중의 bile acid 함량은 HC군에 비해 HRM 및 HRH군에서 2.3배 및 2.7배씩 각각 증가되었다. 분변중의 중성 스테롤류인 cholesterol, coprostanol, coprostanone 함량은 HC군에 비해 다소 높은 경향이었다. 간 microsome의 UDPGT의 활성은 HC군에 비해 HRH군에서 38% 유의적으로 증가되어 가장 높은 활성을 나타내었다. 이상의 결과를 통해 무청은 고콜레스테롤 식이 흰쥐에서 간조직의 UDPGT 활성의 증가로 담즙산의 흡수를 지연시킴으로써 고콜레스테롤 섭취 시 분변중으로 총지질, 총콜레스테롤, 중성지방, 중성스테롤 및 산성스테롤인 담즙산의 배설을 증가시키는 작용을 나타내었고, 따라서 장기능 및 지질대사 개선을 위한 우수 물질임을 알 수 있었다.

EFFECT OF CHRONIC RENAL INSUFFICIENCY ON HEPATIC AND RENAL UDP-GLUCURONYLTRANSFERASES IN RATS

Significant evidence exists regarding altered CYP450 enzymes in chronic renal insufficiency (CRI), although none exists for the phase II enzymes. The objective of this study was to investigate the effect of CRI on hepatic and renal UDP-glucuronyltransferase (UGT) enzymes. Three groups of rats were included: CRI induced by the 5/6th nephrectomy model, control, and control pair-fed (CPF) rats. UGT activities were determined in liver and kidney microsomes by the 3- and 17-glucuronidation of beta-estradiol (E2-3G and E2-17G), glucuronidation of 4-methylumbelliferone (4-MUG), and 3-glucuronidation of morphine (M3G). UGT isoforms responsible for these catalytic activities were screened using recombinant rat UGT1A1, UGT1A2, UGT1A3, UGT1A7, UGT2B2, UGT2B3, and UGT2B8. UGT protein levels were examined by Western blot analysis using polyclonal antibodies. There was no significant difference between CRI and CPF rats in hepatic and/or renal E2-3G (UGT1A1), E2-17G (UGT2B3), 4-MUG (UGT1A6), and M3G (UGT2B1) formation. Formation of E2-17G and 4-MUG in the liver and E2-3G and 4-MUG in the kidney was significantly reduced (p < 0.05) in CPF and CRI rats compared with control rats. The down-regulated glucuronidation activities were accompanied by corresponding reductions in protein content of specific UGT isoforms. These results suggest that CRI does not seem to influence the protein levels or catalytic activity of most of the major hepatic or renal UGT enzymes. The observed down-regulation of hepatic and renal UGTs in CRI and CPF rats could be caused by restricted food intake in these groups of rats.

New approaches to prevent intestinal toxicity of irinotecan-based regimens

Background. Irinotecan is a selective inhibitor of topoisomerase I, an enzyme part of the replication and transcription system of DNA. Irinotecan is employed, with different modalities, in the treatment of metastatic colorectal cancer, and recently it has been officially approved in association with fluorouracil (FU) and leucovorin (LV) as a first-line option in metastatic colorectal cancer. Results. One of the problems linked to the administration of this drug is the high intestinal toxicity, which constitutes its dose limiting toxicity (DLT). In routine practice, loperamide is employed as symptomatic drug for the treatment of CPT-11-induced diarrhoea, but is not completely adequate to control the problem. The role of the intestinal bacterial microflora in the pathogenesis of CPT-11-induced intestinal toxicity has been recently discovered. The active metabolite of CPT-11, SN38, is generated from CPT-11 by sieric carboxylesterase, and subsequently conjugated to SN38-G by hepatic UDP–glucuronyltransferase. SN38-G is the inactive metabolite of CPT-11 and is excreted into the small intestine, from which it is eliminated in the faeces. Some studies have shown the ability of intestinal bacterial β-glucoronidases to transform SN38-G into SN38, causing direct damage to the intestinal mucosa. Thus, alternative strategies such as intestinal alkalinization and anti-cyclooxygenase 2 (COX-2) therapy have been explored. Conclusions. In this review, we will illustrate the mechanisms which cause the CPT-11-induced diarrhoea and the potential measures available to prevent it.

Alterations in hepatic detoxifying enzymes induced by new organophosphorus insecticides following subchronic exposure in rats.

The present study examined the structure-toxicity relationship of two novel phosphorothionates, a methyl ester (RPR-II) and an ethyl ester (RPR-V), with regard to their interaction with certain biochemical indices in rat. Male rats were treated orally with 10% (low), 20% (medium) and 30% (high) doses of the two compounds (14, 28 and 42 microg kg(-1) day (-1) of RPR-II and 33, 66 and 99 microg kg(-1) day(-1) of RPR-V) daily for 90 days. The activity of hepatic glutathione S-transferase (GST) and UDP-glucuronyl transferase (UDPGT) and the level of glutathione (GSH) were estimated at 0, 45 and 90 days of treatment as well as 28 days after cessation of treatment. RPR-II caused statistically significant depletion of GSH after 45 and 90 days of treatment at the high dose, whereas RPR-V depleted GSH only after 90 days at the high dose. RPR-II inhibited GST after 45 and 90 days at medium and high doses, whereas RPR-V caused inhibition of GST after 45 and 90 days only at the high dose. Significantly, UDPGT activity was increased only by the high dose of RPR-II after 90 days. However, a dose and time-dependent increase in UDPGT activity was observed at all three doses of RPR-V after 45 and 90 days. There was no modulation in any of the three indices at low doses of the two organophosphorus insecticides. The withdrawal study revealed that induced changes in hepatic parameters were reversible 28 days after cessation of treatment. The results indicated that the two insecticides had different potential to modulate hepatic GST, UDPGT and GSH due to subchronic exposure and that these metabolic alterations are quite reversible after withdrawal of treatment.

Clinical pharmacokinetics of irinotecan.

This article reviews the clinical pharmacokinetics of a water-soluble analogue of camptothecin, irinotecan [CPT-11 or 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxy-camptoth eci n]. Irinotecan, and its more potent metabolite SN-38 (7- ethyl-10-hydroxy-camptothecin), interfere with mammalian DNA topoisomerase I and cancer cell death appears to result from DNA strand breaks caused by the formation of cleavable complexes. The main clinical adverse effects of irinotecan therapy are neutropenia and diarrhoea. Irinotecan has shown activity in leukaemia, lymphoma and the following cancer sites: colorectum, lung, ovary, cervix, pancreas, stomach and breast. Following the intravenous administration of irinotecan at 100 to 350 mg/m2, mean maximum irinotecan plasma concentrations are within the 1 to 10 mg/L range. Plasma concentrations can be described using a 2- or 3-compartment model with a mean terminal half-life ranging from 5 to 27 hours. The volume of distribution at steady-state (Vss) ranges from 136 to 255 L/m2, and the total body clearance is 8 to 21 L/h/m2. Irinotecan is 65% bound to plasma proteins. The areas under the plasma concentration-time curve (AUC) of both irinotecan and SN-38 increase proportionally to the administered dose, although interpatient variability is important. SN-38 levels achieved in humans are about 100-fold lower than corresponding irinotecan concentrations, but these concentrations are potentially important as SN-38 is 100- to 1000-fold more cytotoxic than the parent compound. SN-38 is 95% bound to plasma proteins. Maximum concentrations of SN-38 are reached about 1 hour after the beginning of a short intravenous infusion. SN-38 plasma decay follows closely that of the parent compound with an apparent terminal half-life ranging from 6 to 30 hours. In human plasma at equilibrium, the irinotecan lactone form accounts for 25 to 30% of the total and SN-38 lactone for 50 to 64%. Irinotecan is extensively metabolised in the liver. The bipiperidinocarbonylxy group of irinotecan is first removed by hydrolysis to yield the corresponding carboxylic acid and SN-38 by carboxyesterase. SN-38 can be converted into SN-38 glucuronide by hepatic UDP-glucuronyltransferase. Another recently identified metabolite is 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxy-camptothecin (APC). This metabolite is a weak inhibitor of KB cell growth and a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). Numerous other unidentified metabolites have been detected in bile and urine. The mean 24-hour irinotecan urinary excretion represents 17 to 25% of the administered dose. Recovery of SN-38 and its glucuronide in urine is low and represents 1 to 3% of the irinotecan dose. Cumulative biliary excretion is 25% for irinotecan, 2% for SN-38 glucuronide and about 1% for SN-38. The pharmacokinetics of irinotecan and SN-38 are not influenced by prior exposure to the parent drug. The AUC of irinotecan and SN-38 correlate significantly with leuco-neutropenia and sometimes with the intensity of diarrhoea. Certain hepatic function parameters have been correlated negatively with irinotecan total body clearance. It was noted that most tumour responses were observed at the highest doses administered in phase I trials, which indicates a dose-response relationship with this drug. In the future, these pharmacokinetic-pharmacodynamic relationships will undoubtedly prove useful in minimising the toxicity and maximise the likelihood of tumour response in patients.

Extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on thyroid hormone turnover in male Sprague-Dawley rats.

Treatment of rats with different polyhalogenated aromatic hydrocarbons strongly decreases plasma T4, with little or no decrease in plasma T3. The extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on thyroid hormone turnover were studied by i.p. administration of a single dose of 10 microg TCDD/kg BW or vehicle (corn oil) to euthyroid (Eu) rats, thyroidectomized (Tx) rats, and Tx rats infused with 1 microg T4 (Tx+T4) or 0.4 microg T3 (Tx+T3)/100 g BW x day by osmotic minipumps. Tx rats showed decreased plasma T4 and T3 and increased plasma TSH levels, decreased hepatic type I deiodinase (D1) and malic enzyme activities, and increased brain type II deiodinase (D2) activities. All parameters were largely restored to Eu levels in Tx+T4 rats and, except for plasma T4 and brain D2 activity, in Tx+T3 rats, validating the thyroid hormone-replaced Tx rats as models to study the peripheral effects of TCDD. Three days after TCDD administration, plasma T4 and free T4 levels were significantly reduced in Eu and Tx+T4 rats, and plasma T3 was significantly reduced in Tx+T3, but not in Eu or Tx+T4 rats. Plasma TSH was not affected by TCDD in any group. Hepatic T4 UDP-glucuronyltransferase (UGT) activity was induced approximately 5-fold by TCDD, whereas T3 UGT activity was only increased by about 20% (P = NS) in the different groups. TCDD produced an insignificant decrease in liver D1 activity in Tx rats and an insignificant increase in brain D2 activity in Tx rats and hormone-replaced Tx rats. Hepatic malic enzyme activity was significantly increased by TCDD in all groups, except Tx rats. These results strongly suggest that the thyroid hormone-decreasing effects of TCDD are predominantly extrathyroidal and mediated by the marked induction of hepatic T4 UGT activity.

Influence of hydrocortisone on the analgesic effect, toxicity and metabolism of aspirin in mice

1. 1. Hydrocortisone (HC; 80 mg/kg body weight, intraperitoneally for 4 days), both alone and in combination with acetylsalicylic acid (ASA; 160 mg/kg body weight, orally, for 4 days), decreased ASA general and specific toxicity via metabolic modulation of drug-metabolizing enzyme systems (intestinal ASA-esterase and hepatic UDP-glucuronyltransferase) and did not change the ASA analgesic effect. 2. 2. ASA alone, given 4 for days, did not change the specific ASA toxicity, but increased its general acute toxicity, which was probably due to alterations in some intestinal and hepatic metabolizing enzyme systems.

Drug Metabolizing Enzymes in the Ostrich ( Struthio camelus): Comparison with the Chicken and the Rat

The activities of selected hepatic and renal drug-metabolizing enzymes of the ostrich, chicken and rats were compared. The concentration of glutathione in the liver and kidneys of the avian species was significantly lower than that in the rat. The activity of ostrich hepatic glutathione S-transferase was 2-fold higher than that of the chicken and the rat and the renal glutathione S-transferase of the ostrich was 10 times higher than that of the rat. The activity of ostrich hepatic UDP-glucuronyl transferase was significantly lower than that of the rat. The activities of hepatic cytochrome P450 1A and 2B1/2 as measured by the dealkylation of ethoxy- and methoxyresorufin, respectively, were higher in the avian species than the rat; no difference was noticed in the activity of aniline hydroxylase. The results show that the activity of ostrich drug-metabolizing enzyme system is quantitatively different from the rat and in many cases also from the chicken.

Gender-specific changes in thyroid hormone-glucuronidating enzymes in rat liver during short-term fasting and long-term food restriction

Glucuronidation is a major pathway of thyroid hormone metabolism in rats, involving at least three different hepatic UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT and androsterone UGT. We have studied the effects of short-term (3 days) fasting and long-term (3 weeks) food restriction to one-third of normal intake (FR33) on hepatic UGT activities for thyroxine (T4), triiodothyronine (T3), bilirubin and androsterone in male and female Wistar rats with either a functional (high activity, HA) or a defective (low activity, LA) androsterone UGT gene. Because food deprivation is known to induce centrally mediated hypothyroidism in rats, results were compared with those obtained in methimazole (MMI)-induced hypothyroid rats. Both fasting and FR33 produced largely parallel increases in T4 and bilirubin UGT activities. These effects were greater in males than in females, and were reproduced in MMI-treated rats. In male and female HA rats, fasting induced insignificant increases in T3 UGT activity and had no effect on androsterone UGT activity. In male HA rats, FR33 was associated with an increase in T3 UGT activity, while androsterone UGT activity showed little change. However, in female HA rats both T3 and androsterone UGT activities were markedly decreased by FR33. Triiodothyronine UGT activity in LA rats was strongly decreased compared with HA rats, but was not further decreased by FR33 in female LA rats, supporting the importance of androsterone UGT for T3 glucuronidation. These results demonstrate different sex-dependent effects of food deprivation on hepatic T4 and T3 glucuronidation that are associated with changes in the expression of bilirubin UGT and androsterone UGT, respectively. For the increased T4 and bilirubin UGT activities at least, these effects appear to be mediated by the hypothyroid state of the (semi)starved animals.

Responses of prepubertal female rats to environmental PCBs with high and low dioxin equivalencies.

An extract of landfill soil containing high levels of polychlorinated biphenyls (PCBs) was refined by alumina-charcoal column chromatography to remove coplanar PCBs and high relative levels of PCDFs. Both extracts were administered ip in corn oil to immature female rats on Days 21 and 22 of age. Rats were terminated on Day 23 and organ weights, enzyme activities, and serum thyroxine (T4) were measured. The change in characteristics caused by charcoal-stripping was confirmed by comparing hepatic microsomal 7-ethoxyresorufin O-deethylase (EROD), 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-dearylase (BROD), and UDP glucuronyl transferase (UDPGT) activities. EROD and 4-nitrophenol (4-NP) UDPGT activities, estimates of aryl hydrocarbon (Ah) receptor agonists present, peaked at 140 mg/kg soil extract, declining at 210 mg/kg; these activities in rats treated with the charcoal-refined soil extract were not induced at low doses, but increased linearly from 70 to 210 mg/kg. Conversely, PROD and BROD activities were modestly induced at 7 to 70 mg/kg of either soil extract; however, at higher doses induction was threefold higher in rats receiving the charcoal-filtered extract. Liver weights increased in a pattern similar to EROD induction. At all doses, the weak uterotropic response was greater in rats receiving the charcoal-filtered extract, probably due to removal of antiestrogenic Ah receptor agonists by the charcoal. Serum total T4 declined similarly in rats receiving either extract to about 40% of control values at the highest doses. Short-term integrated bioassays such as this are useful in detecting multiple complex interactions and can be used to define the net effects of mixtures with changing compositions for improved risk assessment.

Effects of thyroid status and thyrostatic drugs on hepatic glucuronidation of lodothyronines and other substrates in rats

Glucuronidation of iodothyronines in rat liver is catalyzed by at least three UDP-glucuronyltransferases (UGTs): bilirubin UGT, phenol UGT, and androsterone UGT. Bilirubin and phenol UGT activities are regulated by thyroid hormone, but the effect of thyroid status on hepatic glucuronidation of iodothyronines is unknown. We examined the effects of hypothyroidism induced by treatment of rats with propylthiouracil (PTU) or methimazole (MMI) or by thyroidectomy as well as the effects of T4-induced hyperthyroidism on the hepatic UGT activities for T4, T3, bilirubin,p-nitrophenol (PNP), and androsterone. Bilirubin UGT activity was increased in MMI- or PTU-induced hypothyroid and thyroidectomized rats, and decreased in hyperthyroid animals. T4 and, to a lesser extent, T3 UGT activities were increased in MMI- or PTU-induced hypothyroid rats, and T4 but not T3 glucuronidation also showed a significant increase in thyroidectomized rats. T4 but not T3 UGT activity was slightly decreased in hyperthyroid rats. While PNP UGT activity was decreased in thyroidectomized rats and increased in hyperthyroid animals, it was also markedly increased by MMI and slightly increased by PTU-induced hypothyroidism. In T4-substituted rats, MMI did not affect T4, T3, bilirubin and androsterone UGT activities but again strongly induced PNP UGT activity, indicating that this represented a direct induction of PNP UGT by the drug independent of its thyrostatic action. Androsterone UGT activity was hardly affected by thyroid status. Our results suggest a modest, negative control of the hepatic glucuronidation of thyroid hormone by thyroid status, which may be mediated by changes in bilirubin UGT activity. To our knowledge, this is the first report of the marked induction of a hepatic enzyme by MMI, which is not mediated by its thyroid hormone-lowering effect.

Substrate specificity and the use of chitosan-entrapped rabbit hepatic microsomal UDP-glucuronyl transferase for detoxification.

Rabbit hepatic microsomal UDP-Glucuronyl transferase (EC.2.4.1.17) was immobilized in chitosan, which is an ionotropic gelatation agent, and the resulting preparation was used as a biocatalyst for the glucuronidation of toxic substances and drugs such as; 1-naphthol, phenol, 4-nitrophenol, bilirubin, testesterone, estrone, lamotrigine, imipramine and chlorpromazine. For studying the drug metabolism in vitro and obtaining artificial liver support (detoxification) chitosan-entrapped UDPGT towards 1-naphthol was tested by using two model reactor system. The conversion curves of 1-naphthol to its glucuronide indicated that the column reactor system was found to be better and seems suitable for detoxification with a high yield of glucuronide formation.

Preparation and characterization of chitosan-entrapped microsomal UDP-glucuronyl transferase.

The hepatic microsomal UDP-Glucuronyl transferase which catalyze the glucuronidation of drugs, pesticides, carcinogens and other xenobiotics, was immobilized by entrappment in chitosan. Chemical and physical characterization were made by using 1-naphthol as substrate. Thermal, storage and operational stabilities of the immobilized enzyme was also searched and found to be better in comparison with the free enzyme. In conclusion, chitosan gel beads appear to have good characteristics for use as UDPGT immobilization support suitable for large scale use and offer the prospect that immobilized UDPGT may become an important form of catalyst in medicine.

Effect of dairy products on initiation of precursor lesions of colon cancer in rats

This study reports the modulating effect of some dairy products on initiation of putative preneoplasic lesions in rat colon (aberrant crypts) by 1,2-dimethylhydrazine dihydrochloride. Uninoculated skim milk, skim milk fermented with Bifidobacterium sp Bio (Danone strain 173010), and a suspension of the same lactic acid bacteria were incorporated in the animals' diet. The tested diets significantly reduced the incidence of aberrant crypts compared with the control diet by 51%, 49%, and 61%, respectively. The effects of the diets on cecal pH, hepatic UDP-glucuronyltransferase activity, and cecal microflora enzyme beta-glucuronidase were also studied. There was no significant difference in cecal pH between rats fed experimental diets and control rat. The diet supplemented with the Bifidobacterium strain suspension significantly decreased only the cecal beta-glucuronidase activity. Both enzyme activities were reduced in rats fed fermented skim milk- or uninoculated skim milk-supplemented diets compared with control animals.

Hormonal disregulation mechanism in the rat thyroid tumor induced by diniconazole.

To assess the toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diniconazole, (E)-1-(2,4-dichlorophenyl)- 4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol, a 3-month subacute feeding study was conducted in male Crj: CD (SD) rats by administering diniconazole in diet at concentrations of 0, 100, 1,000, or 2,000 ppm. Examinations mainly for thyroid functions were performed at Weeks 2, 4 and 13. Measurement of serum hormone levels revealed continuous decreases in serum thyroxine (T4) and free T4 levels at and above 1,000 ppm and increase in serum thyroid stimulating hormone (TSH) level at 2,000 ppm concurrently with liver weight and hepatic UDP-glucuronyltransferase (UDP-GT) increases at and above 1,000 ppm. No changes were observed in serum triiodothyronine (T3) and free T3 levels. Increase in thyroid uptake of 125I and organification of 125I in the thyroid at 2,000 ppm and thyroid follicular cell hyperplasia at and above 1,000 ppm were also observed. However, no compound-related changes were observed in autopsy and organ weight in the thyroid. Based on the above results, diniconazole induces increases in the hepatic UDP-GT activity and the thyroid hormone excretion from the liver. The increased excretion of thyroid hormones causes decrease in serum T4 and free T4 levels, triggering the feedback mechanism of the pituitary gland, promotion of TSH release from the pituitary gland and increase in serum TSH level. The increased serum TSH level probably leads to increased 125I uptake of thyroid and thyroid follicular cell hyperplasia. Thus, the thyroid tumorigenesis in rats treated with diniconazole is due to the secondary overstimulant effect on the thyroid by increased serum TSH level. The toxicological significance in humans is extremely low and it is unlikely that diniconazole would increase thyroid tumor in humans even if diniconazole were to alter normal thyroid hormone level in humans.

Effects of diethofencarb on thyroid function and hepatic UDP-glucuronyltransferase activity in rats.

To examine the mechanism and toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diethofencarb (isopropyl 3,4-diethoxycarbanilate), male Sprague-Dawley rats were fed diethofencarb in diets at concentrations of 0, 5,000 or 20,000 ppm for 3 months. Examinations mainly for thyroid functions including thyroid uptake of 125I, serum thyroid hormone and thyroid stimulating hormone (TSH) level, hepatic UDP-glucuronyltransferase (UDP-GT) activity and histopathological examination in thyroid were performed at week 13. Decreases of body weights and food consumptions were observed at and above 5,000 ppm. Under these conditions, decrease of serum free T4 and increase of serum TSH level were observed only at 20,000 ppm, concurrently with liver weight increase at and above 5,000 ppm and increase of hepatic UDP-GT activity at 20,000 ppm. However, no compound related effects were noted in thyroid weight, thyroid uptake of 125I and gross or histopathological examination in thyroid. These results indicate that the administration of diethofencarb leads to an increase in UDP-GT activity and acceleration of thyroid hormone excretion from the liver. The acceleration causes a decrease in serum free T4 level, triggering the feedback mechanism of the pituitary gland, promotion of TSH release and consequently an increase in serum TSH level. Thus, the slightly higher incidence of thyroid follicular cell tumors observed in the chronic and oncogenicity study with non-genotoxic diethofencarb is considered to be caused by these weak pituitary-thyroid hormonal imbalances. The toxicological significance in humans is extremely low according to the well established facts that the chronic TSH stimulating would not induce thyroid tumors in humans and humans may be less sensitive than rats in regard to the response to goitrogenic stimuli.