Investigation of the potential of conjugated linoleic acid (Cla) to cause peroxisome proliferation in rats.

Conjugated linoleic acid (CLA) is reported as having several beneficial effects including anticarcinogenic, cholesterol-lowering and anti-atherogenic properties; however, CLA has also been reported as a putative peroxisome proliferator in mice. In this study the ability of CLA to cause peroxisome proliferation in the rat, as measured by accepted enzyme markers was investigated. Male Wistar rats were fed a semi-purified diet containing 0.0, 1.5 or 5.0 energy % CLA for 4 weeks. A positive control group were given 250 mg clofibrate/kg by gavage for 4 days. Hepatic cyanide-insensitive palmitoyl coenzyme A (PCoA) oxidase and carnitine acetyl transferase (CAT) activities and total cytochrome P450 (CYP) levels were measured. CLA had no effect on body weight or liver/body weight ratios, but clofibrate significantly increased mean liver/body weight ratio by 41.6%. Clofibrate-treated rats showed typical changes with increases in hepatic PCoA oxidase and CAT activity (5.8-fold and 22.8-fold) and in total CYP (1.66-fold) compared with control. There were no differences between the control group and the groups fed CLA for either the peroxisomal enzymes or total CYP. These results suggest that CLA does not act in the rat as a classical peroxisome proliferator and that there may be a species difference in the effects on rat and mice.     

Modulation of sulphobromophthalein excretion by ethacrynic acid.

1. Ethacrynic acid (EA), a phenoxyacetic acid diuretic, has similar effects to tienilic acid (TA) on rat liver glutathione S-transferase (GST) activity in vitro, using either 1-chloro-2,4-dinitrobenzene or sulphobromophthalein (BSP) as a substrate. EA inhibits the basic rat liver GST, with inhibition being greater with GST containing subunits 3 and 4 than with those containing subunits 1 and 2. 2. In vitro inhibitors of GST can inhibit biliary excretion of BSP in a perfused liver. 3. A single bolus dose of EA had no effect on BSP excretion from the isolated perfused rat liver, and this is most likely due to the rapid disappearance of EA from the perfusion media. Experiments using perfused rat liver indicated that a sustained high concentration of EA in the perfusion media has an inhibitory effect on the excretion of both unchanged and conjugated BSP. 4. A decrease in BSP excretion may not be an indicator of liver damage, but a consequence of GST inhibition.     

Studies of early hepatocellular proliferation and peroxisomal proliferation in Sprague-Dawley rats treated with tumorigenic doses of clofibrate.

Clofibrate, a peroxisome proliferator, is hepatocarcinogenic in rats in a dose-dependent fashion. While there is a relationship between peroxisome proliferation and rodent liver carcinogenesis, recent evidence also suggests an association between the tumorigenicity of peroxisome proliferators and sustained cell proliferation. To investigate the role of early cell proliferation in clofibrate-induced carcinogenesis and the predictive potential of this endpoint, in a 3-month study, rats were fed clofibrate doses equivalent to those used in the chronic bioassay, and cell proliferation was determined after 1 week and 3 months, using a 1-week continuous bromodeoxyuridine (BrdU)-labeling technique. Adult Sprague-Dawley rats were fed clofibrate at 1500, 4500, or 9000 ppm. Six rats/sex/group were killed after 1 or 13 weeks of treatment. Osmotic minipumps containing BrdU were implanted into rats 7 days prior to necropsy to determine the cumulative 7-day hepatocyte labeling index immunohistochemically. A dose-related increase in hepatocyte labeling index was seen after 1 week of treatment. However, at 13 weeks, sustained increases in hepatocyte proliferation were not seen; but a dose-related decrease in the hepatocyte labeling index was observed. Liver stereology at 13 weeks demonstrated a dose-related increase in liver weight and volume, but a decrease in hepatocyte nuclei per unit volume, a minimal increase or no change in the total number of hepatocyte nuclei per liver, and an absolute decline in the total number of BrdU-labeled hepatocyte nuclei per liver. These data suggest that in rats, clofibrate may influence hepatocarcinogenicity by decreases in normal hepatocyte proliferation over time and this effect may influence the pathogenesis of tumors at time points beyond 13 weeks of treatment.     

Inhibition of liver glutathione S-transferase activity in rats by hypolipidemic drugs related or unrelated to clofibrate

The effects of in vivo administration of six hypolipidemic drugs on rat liver glutathione S-transferase activity were compared. This activity was measured with sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) or 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Except for the nicotinic acid derivative ethanolamine oxiniacate, all the compounds tested significantly reduced it, whether or not they were related to clofibrate. The hepatic glutathione concentration either remained unchanged or only increased slightly after treatment with the various drugs. When measured, the maximal excretion rate of bile BSP dropped significantly, but not that of phenol-3,6-dibromophthalein (DBSP). Hepatic dye uptake and storage were not impaired. These results show that hypolipidemic drugs of the peroxisome proliferator type inhibit rat liver glutathione S-transferase activity and may reduce transport of anions conjugated with glutathione before excretion.     

Apparent induction of microsomal carboxylesterase activities in tissues of clofibrate-fed mice and rats

Treatment with 0.5% (w/w) dietary clofibrate, a peroxisome proliferator, for 14 days induced microsomal carboxylesterase activities for five substrates including malathion, clofibrate, diethylsuccinate, diethylphthalate, and p-nitrophenylacetate in liver and kidney of male Swiss-Webster mice and Sprague-Dawley rats. The induction was substrate, tissue, and species dependent. The carboxylesterase activity was induced in mouse from 1.2- to 2.2-fold (liver) and from 1.1- to 1.7-fold (kidney) depending upon substrate used. Analogous values from rat ranged from 1.0- to 1.4-fold (liver) and from 1.1- to 1.8-fold (kidney). Enzyme activities were either decreased or not affected in testes of treated mice and rats. Substituted trifluoroketones ("transition-state" inhibitors of carboxylesterase) were found to be very potent inhibitors of clofibrate-metabolizing carboxylesterase(s) and to be potentially useful in distinguishing among isozymes. The inhibition data suggested that changes in carboxylesterase activity following clofibrate treatment were both qualitative and quantitative.     

Toxicological studies on a benzofurane derivative. II. Demonstration of peroxisome proliferation in rat liver

The uricosuric drug benzbromarone (3,5-dibromo-4-hydroxyphenyl)-1-(2-ethyl-3-benzofuranyl)methanone, a benzofurane derivative, was studied for its effects on parameters related to hepatic peroxisome proliferation. Groups of male F-344 rats were fed either basal diet, the peroxisome proliferator clofibrate at 5000 ppm as a comparison compound, or benzbromarone at two doses, 1000 and 2000 ppm. Benzbromarone and clofibrate produced hepatomegaly and increases in the activities of catalase, acyl CoA oxidase, malate dehydrogenase, and glycerol-3-phosphate dehydrogenase. Benzbromarone and clofibrate also both induced similar histologic and ultrastructural changes in hepatocytes, including induction of peroxisomes. Therefore, benzbromarone acted as a peroxisome-proliferating agent in rats under these conditions. Benzbromarone differs from other peroxisome proliferators in its chemical structure, uricosuric action, and the morphology of liver peroxisomes that were induced by exposure.     

Mechanisms involved in growth inhibition induced by clofibrate in hepatoma cells

Low concentrations of some peroxisome proliferators have been found to decrease apoptosis in rat liver cells, whereas higher but pharmacological concentrations have been found to inhibit cell proliferation or to induce apoptosis in human and rat hepatoma cells. The highly deviated JM2 rat hepatoma cell line was used to examine the mechanisms underlying the inhibitory effect on cell proliferation. Clofibrate chiefly inhibited cell proliferation in these cells. Parallel to the decrease in cell proliferation there was an increase of peroxisome proliferator activated receptor (PPAR) gamma and of protein phosphatase 2A, whose importance was confirmed, respectively, by using antisense oliginucleotides (AS-ODN) or okadaic acid. The increase of protein phosphatase 2A induced by PPARgamma caused a decrease of MAPK, an intracellular signaling transduction pathway, as shown by evaluation of Erk1,2 and c-myc. In light of these results, clofibrate, like conventional synthetic ligands of PPARgamma, may be regarded as a possible prototype anti-tumour drug.     

Effect of chlorophenoxyacetic acid herbicides on glycine conjugation of benzoic acid.

1. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) (0.1-0.5 mmol/kg i.p.) delayed the disappearance of injected benzoate from blood and diminished the urinary excretion of the formed benzoylglycine, but elevated the blood levels of benzoylglycine in rat, suggesting that these herbicides interfere with both the formation and the renal transport of benzoylglycine. 2. Inhibition of the renal excretion of benzoylglycine by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in rat injected with benzoylglycine. 3. Inhibition of benzoylglycine formation from benzoic acid by 2,4-D or 2,4,5-T (0.5 mmol/kg i.p.) was directly demonstrated in renal pedicles-ligated rats injected with benzoate. 4. Neither 2,4-D nor 2,4,5-T influenced the hepatic concentrations of ATP, coenzyme A (CoA) or glycine; therefore, it is unlikely that they inhibit glycine conjugation of benzoic acid by diminishing the availability of co-substrates. 5. Although the chlorophenoxyacetic acids did not appear to be a substrate for the mitochondrial acyl-CoA synthetases, both 2,4-D and 2,4,5-T diminished the activity of benzoyl-CoA synthetase (but not that of benzoyl-CoA:glycine N-acyltransferase) in solubilized hepatic mitochondria. These findings suggest that 2,4-D and 2,4,5-T impair benzoylglycine formation in rat by inhibiting benzoyl-CoA synthetase.     

Effects of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid on peroxisomal enzymes in rat liver

The effects of feeding 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on the level of peroxisomal enzymes in rat liver were studied. The concentration of triglyceride in serum was decreased and the activity of cyanide-insensitive palmitoyl-CoA oxidation, catalase and carnitine acetyltransferase increased. However, the extent of the increase in the activity of these enzymes by treatment with 2,4-D was less pronounced than that by 2,4,5-T treatment. The administration of 2,4-D or 2,4,5-T increased the concentration of polypeptide with a mol. wt of 80,000 in the light mitochondrial fractions of the liver from the rats.     

Effects of in vivo treatment with a new fluorinated macrolide (P-0501A) and other erythromycins on drug clearance and hepatic functions in perfused rat liver

The hepatic clearance and the effects of a new fluorinated macrolide (P-0501A) on the functions of the isolated, perfused rat liver were compared with two known erythromycins--the base and the estolate--after 7 days of treatment (1.36 mmol/kg po daily). The in vitro metabolism of the antibiotics was induced to different extent but only the base and P-0501A were cleared from the perfusate and the liver faster than in untreated animals. In untreated rats the therapeutically active form of P-0501A was excreted in the bile more than the base and the estolate; after pretreatment, biliary excretion of all erythromycins was nearly double. The content of inactive, complexed cytochrome P-450 was increased only by the base and estolate, with various effects on microsomal activities (some induced, e.g. aminopyrine demethylation, other reduced, e.g. pentobarbital clearance). The clearance and biliary excretion of sulphobromophthalein was not affected by treatment with P-0501A or the base, but was significantly reduced by estolate.     

In vitro uptake of organic ions by renal cortical tissue of rats treated acutely with 2,4,5-trichlorophenoxyacetic acid.

The effect of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on renal cortical function has been studied in male adult rats. Significant decreases in organic acid and organic base transport were measured when rats were pretreated with 2,4,5-T 24 hr before in vitro analysis of renal function. Experiments in which injections of p-aminohippurate were given showed no effect on organic acid or organic base transport by renal cortical slices. The data are interpreted to mean that pretreatment with 2,4,5-T has a depressive influence on the transport of some organic ions. Adult animals which were treated daily with 2,4,5-T accumulated a large body store of this herbicide during the first 6 days of administration. However, by 9 days the animals excreted essentially the dose of 2,4,5-T administered, and by 16 days the herbicide accumulated during the first 6 days had been almost totally eliminated. This chronic excretion pattern may explain why only moderate toxicity has been reported for this herbicide.     

The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to rats and dogs

Clearance of ¹⁴C activity from the plasma and its elimination from the body of rats and dogs were determined after single oral doses of [carboxy-¹⁴C]2,4,5-T. The half-life values for the clearance of ¹⁴C activity from the plasma of rats given doses of 5, 50, 100 or 200 mg/kg were 4.7, 4.2, 19.4 and 25.2 hr, respectively; half-lives for elimination from the body were 13.6, 13.1, 19.3 and 28.9 hr, respectively. The apparent volume of distribution also increased with dose. Urinary excretion of unchanged 2,4,5-T accounted for most of the ¹⁴C activity eliminated from the body of rats. A small amount of unidentified metabolite was detected in the urine when rats were given 100 or 200 mg/kg but not 5 or 50 mg/kg. These results show that the distribution, metabolism and excretion of 2,4,5-T are markedly altered when large doses are administered.In dogs given 5 mg/kg, the half-life values for clearance from plasma and elimination from the body were 77.0 and 86.6 hr, respectively, offering a plausible explanation of why 2,4,5-T is more toxic in dogs than in rats. Appreciable excretion in the feces was noted and three unidentified metabolites were detected in urine of dogs, indicating a considerable difference in metabolism of 2,4,5-T by dogs and rats given the same dose.     

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation (r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal epoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal beta-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.     

Effect of clofibrate on plasma lipid concentration and liver malic enzyme gene expression in rats with experimental chronic renal failure

Fibrates have been used clinically to treat dyslipidemias, including chronic renal failure (CRF)-related hypertriacylgliceridemia. In addition to their effects on plasma triacylglycerol concentration, fibrates also induce hepatomegaly (due to peroxisome proliferation) and increase liver malic enzyme activity. Since most experiments regarding fibrates action have been performed on healthy animals, in this paper we compare the effect of clofibrate on: a) plasma lipid concentration; b) liver weight; c) liver malic enzyme gene expression (malic enzyme activity, malic enzyme protein level and malic enzyme mRNA abundance) in control (sham-operated) animals and rats with CRF. The data presented in this paper indicate that: a) the clofibrate treatment causes a decrease in triacylglycerol concentration both in the control and rats with CRF, however the effect of the drug was more pronounced in the latter; b) administration of clofibrate induces hepatomegaly both in the control and rats with CRF; c) the liver malic enzyme gene expression is similarly affected by clofibrate in the control and rats with CRF. It is concluded that the beneficial, therapeutic effect of clofibrate on plasma lipid concentration is more pronounced in rats with CRF, but the side effects (hepatomegaly and the increase in malic enzyme gene expression) of fibrates are essentially similar in the control and rats with CRF.     

壳聚糖复方制剂对脂肪肝大鼠的治疗作用及其机制的探讨

目的 :进一步研究壳聚糖复方制剂对实验性脂肪肝大鼠的治疗作用及其机制。方法 :采用小剂量CCl4 致肝损伤并高脂饮食建立大鼠脂肪肝模型 ,给予模型大鼠壳聚糖复方制剂 0 .7g·kg- 1·d- 1灌服 8wk ,检测肝脏三酰甘油 (TG )、总胆固醇(TC)、血清及肝脏游离脂肪酸 (FFA)含量 ,检测肝脂变面积及肝脏过氧化物酶体增殖物激活受体α(PPARα)mRNA的表达情况。结果 :CCl4 损伤并高脂饮食可引起实验性大鼠肝脏明显脂肪变性 ,肝脏TG ,TC ,FFA及血清FFA含量升高 ,肝细胞PPARαmRNA表达减少。与模型组比较 ,壳聚糖复方制剂能明显降低肝脏TG ,TC ,FFA及血清FFA含量(P <0 .0 1) ,提高肝细胞PPARαmRNA的表达(P <0 .0 1)。结论 :壳聚糖复方制剂对实验性脂肪肝大鼠具有一定的治疗作用 ,其作用机制可能与其诱导PPARα表达 ,促进肝脏摄取、氧化脂肪酸有关。     

Tissue specific basal expression of soluble murine epoxide hydrolase and effects of clofibrate on the mRNA levels in extrahepatic tissues and liver

 The soluble epoxide hydrolase mRNA level in liver was increased eight-fold upon administration of the hypolipidemic drug and peroxisome proliferator clofibrate for 7 days to mice. The soluble epoxide hydrolase mRNA was back at control levels within 1–2 days after clofibrate withdrawal. The highest expression was in liver, intestine and kidney. Lower levels were found in heart and muscle and very low levels were found in testes, lung, brain and spleen. The mRNA levels were increased in liver, kidney and heart by clofibrate. Received: 17 January 1995/Accepted: 12 June 1995     

Effects of Quinuronium Sulfate Pre-exposure on Hepatic Excretion of Sulfobromophtalein (BSP) as Measured by Isolated Perfused Rat Liver

Abstract: Effects of quinuronium sulphate pre-exposure on hepatic excretion of sulfobromophthalein (BSP) as measured by isolated perfused rat liver was investigated. Seventeen rats were used in the study. Rats were divided into controls (Group 1, n = 5), low quinuronium treated animals (Group 2, n = 6), and high quinuronium treated animals (Group 3, n = 6) receiving 0, 1.5 or 2.3 mg quinuronium sulphate/kg intraperitoneally, respectively. Each animal was dosed 30 min. before sacrifice. After 30 min. of equilibrium perfusion, BSP was introduced and the experiment continued for 2 hours. Perfusate and bile were collected at designated time intervals and the volume of bile was noted. BSP in perfusate and bile was assayed. Half-life in minutes (t_1/2$pLS.E.) of BSP disappearance from perfusate in groups 1, 2 and 3 were 12.5$pL1.8, 13.7$pL1.0 and 16.8$pL3.3, respectively. Group 2 and 3 rats had a significant decrease of BSP excretion in bile (P<0.01) compared to group 1 rats. Bile volumes decreased in both quinuronium pre-exposed groups of rats compared to control rats. These observations indicate that quinuronium affects liver function.     

Pharmacokinetic modulation of irinotecan metabolites by sulphobromophthalein in rats

The purpose of this study was to modulate the pharmacokinetics of irinotecan metabolites, SN-38 and SN-38-glucuronide, by possibly reducing biliary excretion, which in turn could lower irinotecan toxicity. SN-38-glucuronide is associated with severe diarrhoea that occurs after irinotecan therapy as a result of enteric injury caused by SN-38. Sulphobromophthalein is used clinically as a drug for testing liver function and is considered to be a safe drug. We investigated the effect of sulphobromophthalein on the disposition of irinotecan metabolites after CPT-11 (7-ethyl-10-[10-4-(1-piperidino)-1-piperidino]-carbonyloxy-camptothecin) administration. Wistar rats were administered CPT11 (500 microg/body) in saline as a bolus injection into the femoral vein through a catheter. The volume of drug solution injected into each animal was 1 mL. Rats were either administered CPT-11 alone or simultaneously with sulphobromophthalein (20 mg/body). After administration, blood and bile samples were taken at appropriate intervals and analysed by HPLC. Co-administration of sulphobromophthalein partially inhibited the biliary excretion of SN-38-glucuronide with a concomitant increase in its area under the plasma concentration-time curve (AUC) but did not significantly alter the biliary excretion and AUC of the active metabolite SN-38. These results suggested that cotreatment of CPT-11 with sulphobromophthalein might decrease intraluminal SN-38 concentrations without altering the pharmacokinetics of SN-38.     

Differential effects of dehydroepiandrosterone and clofibrate on the binding of 7,12-dimethyl benz(a)anthracene to hepatic DNA in vivo--a preliminary study

The effects of feeding two compounds, dehydroepiandrosterone (DHEA), an adrenal steroid, and clofibrate (CLOF) to rats (which are both hypolipidemic, hepatomegaly inducing and hepatic peroxisome proliferating agents) on the binding of 7,12-dimethylbenz(a)anthracene (DMBA) to hepatic DNA in vivo is compared. Male Sprague Dawley rats (two-three months old) were fed either DHEA or CLOF for 14 days at a dietary level of 0.8%. Control rats were pair fed. An increase in liver weight followed by increases per whole liver in total protein, without much change in DNA content was observed. Subsequently, all the animals were given a single intraperitoneal dose of [3H]DMBA (133 mumol/kg body weight, 102 microCi/rat) in 250 microliters dimethyl sulfoxide. Forty-eight hours later, binding of DMBA to hepatic DNA was determined. The results indicate that DMBA binding to DNA was reduced by 67% in DHEA-fed rats whereas in clofibrate-fed rats it was not significantly different from that of the controls.     

Mirex-induced suppression of biliary excretion of polychlorinated biphenyl compounds

Isolated perfused rat liver preparations were utilized to study the drug-induced modification of biliary excretion of [¹⁴C]4-chlorobiphenyl (1-CB) and [¹⁴C]2,4,5-2′,4′,5′-hexachlorobiphenyl (6-CB) and their metabolites. The effect of pretreatment of rats with mirex was compared with that of phenobarbital (PB) and 6-CB by measuring the biliary excretion of 1-CB and the pharmacokinetics of 1-CB and its metabolites in the perfusate of liver preparations obtained from control and treated rats. The biliary excretion of 1-CB metabolites by mirex-pretreated livers was suppressed by 92% of that in control livers. The rate of metabolism of 1-CB by mirexpretreated livers was slightly decreased. However, the suppression of biliary excretion of 1-CB metabolites does not appear to be related either to decreased rate of metabolism or to the rate of bile flow. These conclusions are borne out from the following two observations: first, the metabolites of 1-CB accumulate at increasing concentrations in the perfusate of mirexpretreated livers; secondly, mirex-pretreated livers had elevated bile flows. Biliary excretion of exogenously added metabolites of 1-CB by mirextreated livers was suppressed by 85% of that of control livers. PB and 6-CB pretreatment caused a statistically significant increase in the biliary excretion of 1-CB metabolites. This was accompanied by a slight but statistically nonsignificant increase in the rate of bile flow. Biliary excretion of 6-CB was completely suppressed by mirex pretreatment and was unaffected by PB. These results indicate that while PB-induced modification of biliary excretion of PCBs may be associated with their metabolism, mirex-induced suppression is associated with the transport of otherwise readily excretable metabolites from the hepatocytes into the bile canaliculi.