Deletion of NADPH oxidase 2 in chondrocytes exacerbates ethanol-mediated growth plate disruption in mice without major effects on bone architecture or gene expression.

BACKGROUND: Excess reactive oxygen species generated by NADPH oxidase 2 (Nox2) in response to ethanol exposure mediate aspects of skeletal toxicity including increased osteoclast differentiation and activity. Because perturbation of chondrocyte differentiation in the growth plate by ethanol could be prevented by dietary antioxidants, we hypothesized that Nox2 in the growth plate was involved in ethanol-associated reductions in longitudinal bone growth. METHODS: Nox2 conditional knockout mice were generated, where the essential catalytic subunit of Nox2, cytochrome B-245 beta chain (Cybb), is deleted in chondrocytes using a Cre-Lox model with Cre expressed from the collagen 2a1 promoter (Col2a1-Cre). Wild-type and Cre-Lox mice were fed an ethanol Lieber-DeCarli-based diet or pair-fed a control diet for 8 weeks. RESULTS: Ethanol treatment significantly reduced the number of proliferating chondrocytes in the growth plate, enhanced bone marrow adiposity, shortened femurs, reduced body length, reduced cortical bone volume, and decreased mRNA levels of a number of osteoblast and chondrocyte genes. Conditional knockout of Nox2 enzymatic activity in chondrocytes did not consistently prevent any ethanol effects. Rather, knockout mice had fewer proliferating chondrocytes than wild-type mice in both the ethanol- and control-fed animals. Additional analysis of tibia samples from Nox4 knockout mice showed that loss of Nox4 activity also reduced the number of proliferating chondrocytes and altered chondrocyte size in the growth plate. CONCLUSIONS: Although Nox enzymatic activity regulates growth plate development, ethanol-associated disruption of the growth plate morphology is independent of ethanol-mediated increases in Nox2 activity.

Diet Supplementation with Soy Protein Isolate, but Not the Isoflavone Genistein, Protects Against Alcohol-Induced Tumor Progression in DEN-Treated Male Mice.

Diethylnitrosamine-treated male mice were assigned to 4 groups: a casein-based 35% high fat ethanol liquid diet (EtOH), an EtOH diet made with soy protein isolate protein (EtOH/SOY), an EtOH liquid diet supplemented with genistein (EtOH/GEN) and a chow group. EtOH feeding, final concentration 5% (v/v), continued for 16 wks. EtOH increased incidence and multiplicity of basophilic lesions and adenomas compared to the chow group, (p < 0.05). The EtOH/SOY group had reduced adenoma progression when compared to the EtOH and EtOH/GEN group, (p < 0.05). Genistein supplementation had no protective effect. Soy feeding significantly reduced serum ALT concentrations (p < 0.05), decreased hepatic TNFα and CD-14 expression and decreased nuclear accumulation of NFκB protein in EtOH/SOY-treated mice compared to the EtOH group (p < 0.05). With respect to ceramides, high resolution MALDI-FTICR Imaging mass spectrometry revealed changes in the accumulation of long acyl chain ceramide species, in particular C18, in the EtOH group when compared to the EtOH/SOY group. Additionally, expression of acid ceramidase and sphingosine kinase 1 which degrade ceramide into sphingosine and convert sphingosine to sphingosine-1-phosphate (S1P) respectively and expression of S1P receptors S1PR2 and S1PR3 were all upregulated by EtOH and suppressed in the EtOH/SOY group, p < 0.05. EtOH feeding also increased hepatocyte proliferation and mRNA expression of ß-catenin targets, including cyclin D1, MMP7 and glutamine synthase, which were reduced in the EtOH/SOY group, p < 0.05. These findings suggest that soy prevents tumorigenesis by reducing inflammation and by reducing hepatocyte proliferation through inhibition of EtOH-mediated ß-catenin signaling. These mechanisms may involve blockade of sphingolipid signaling.

Alcohol consumption, Wnt/ß-catenin signaling, and hepatocarcinogenesis.

Alcohol is a well-established risk factor for hepatocellular carcinoma, and the mechanisms by which alcohol liver cancer is complex. It has been suggested that ethanol (EtOH) metabolism may enhance tumor progression by increasing hepatocyte proliferation. To test this hypothesis, ethanol (EtOH) feeding of male mice began 7 weeks post-injection of the chemical carcinogen diethylnitrosamine (DEN), and continued for 16 weeks, with a final EtOH concentration of 28% of total calories. As expected, EtOH increased the total number of cancerous foci and liver tumors identified in situ fixed livers from the EtOH+DEN group compared to corresponding pair-fed (PF)+DEN and chow+DEN control groups. In the EtOH+DEN group, tumor multiplicity corresponded to a 3- to 4-fold increase in proliferation and immunohistochemical staining of ß-catenin in non-tumorigenic hepatocytes when compared to the PF+DEN and chow+DEN groups, p<0.05. Analysis of EtOH-treated livers from a previously published rat model of chronic liver disease revealed increases in hepatocyte proliferation accompanied by a hepatic depletion of retinol and retinoic acid stores (p<0.05), nuclear accumulation of ß-catenin (p<0.05), increased cytosolic expression p-GSK3ß (p<0.05), significant upregulation of soluble Wnts, Wnt2, and Wnt7a, and increased expression of several ß-catenin targets involved in tumor promotion and progression, cyclin D1, c-myc, WISP1, and MMP7 (p<0.05). These data suggest that chronic EtOH consumption activates the Wnt/ß-catenin signaling pathway, which increases hepatocyte proliferation thus promoting tumorigenesis following an initiating insult in the liver.

Dietary fat source alters hepatic gene expression profile and determines the type of liver pathology in rats overfed via total enteral nutrition.

To determine if dietary fat composition affects the progression of nonalcoholic fatty liver disease (NAFLD), we overfed male Sprague-Dawley rats low (5%) or high (70%) fat diets with different fat sources: olive oil (OO), corn oil (CO), or echium oil (EO), with total enteral nutrition (TEN) for 21 days. Overfeeding of the 5% CO or 5% EO diets resulted in less steatosis than 5% OO (P < 0.05). Affymetrix array analysis revealed significant differences in hepatic gene expression signatures associated with greater fatty acid synthesis, ChREBP, and SREBP-1c signaling and increased fatty acid transport (P < 0.05) in the 5% OO compared with 5% CO group. The OO groups had macrosteatosis, but no evidence of oxidative stress or necrosis. The 70% CO and 70% EO groups had a mixture of micro- and macrosteatosis or only microsteatosis, respectively; increased oxidative stress; and increased necrotic injury relative to their respective 5% groups (P < 0.05). Oxidative stress and necrosis correlated with increasing peroxidizability of the accumulated triglycerides. Affymetrix array analysis comparing the 70% OO and 70% CO groups revealed increased antioxidant pathways and lower expression of genes linked to inflammation and fibrosis in the 70% OO group. A second study in which 70% OO diet was overfed for 50 days produced no evidence of progression of injury beyond simple steatosis. These data suggest that dietary fat type strongly influences the progression of NAFLD and that a Mediterranean diet high in olive oil may reduce the risk of NAFLD progressing to nonalcoholic steatohepatitis.

Soy protein isolate reduces hepatosteatosis in yellow Avy/a mice without altering coat color phenotype.

Agouti (A(vy)/a) mice fed an AIN-93G diet containing the soy isoflavone genistein (GEN) prior to and during pregnancy were reported to shift coat color and body composition phenotypes from obese-yellow towards lean pseudoagouti, suggesting epigenetic programming. Human consumption of purified GEN is rare and soy protein is the primary source of GEN. Virgin a/a female and A(vy)/a male mice were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) (the same approximate GEN levels as in the above mentioned study) for 2 wks prior to mating. A(vy)/a offspring were weaned to the same diets and studied at age 75 d. Coat color distribution did not differ among diets, but SPI-fed, obese A(vy)/a offspring had lower hepatosteatosis (P < 0.05) and increased (P < 0.05) expression of CYP4a 14, a PPARalpha-regulated gene compared to CAS controls. Similarly, weanling male Sprague-Dawley (SD) rats fed SPI had elevated hepatic Acyl Co-A Oxidase (ACO) mRNA levels and increased in vitro binding of PPARalpha to the PPRE promoter response element. In another hepatosteatosis model, adult SD rats fed a high fat/cholesterol diet, SPI reduced (P < 0.05) steatosis. Thus, 1) consumption of diets made with SPI partially protected against hepatosteatosis in yellow mice and in SD rats, and this may involve induction of PPARalpha-regulated genes; and 2) the lifetime (in utero, neonatal and adult) exposure to dietary soy protein did not result in a shift in coat color phenotype of A(vy)/a mice. These findings, when compared with those of previously published studies of A(vy)/a mice, lead us to conclude that: 1) the effects of purified GEN differ from those of SPI when GEN equivalents are closely matched; 2) SPI does not epigenetically regulate the agouti locus to shift the coat color phenotype in the same fashion as GEN alone; and 3) SPI may be beneficial in management of non-alcoholic fatty liver disease.

Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes.

Increase dincidences of hepatotoxicity have been observed in diabetic patients receiving drug therapies. Neither the mechanisms nor the predisposing factors underlying hepatotoxicity in diabetics are clearly understood. Animal studies designed to examine the mechanisms of diabetes-modulated hepatotoxicity have traditionally focused only on bioactivation/detoxification of drugs and toxicants. It is becoming clear that once injury is initiated, additional events determine the final outcome of liver injury. Foremost among them are two leading mechanisms: first, biochemical mechanisms that lead to progression or regression of injury; and second, whether or not timely and adequate liver tissue repair occurs to mitigate injury and restore liver function. The liver has a remarkable ability to repair and restore its structure and function after physical or chemical-induced damage. The dynamic interaction between biotransformation-based liver injury and compensatory tissue repair plays a pivotal role in determining the ultimate outcome of hepatotoxicity initiated by drugs or toxicants. In this review, mechanisms underlying altered hepatotoxicity in diabetes with emphasis on both altered bioactivation and liver tissue repair are discussed. Animal models of both marked sensitivity (diabetic rats) and equally marked protection (diabetic mice) from drug-induced hepatotoxicity are described. These examples represent a remarkable species difference. Availability of the rodent diabetic models offers a unique opportunity to uncover mechanisms of clinical interest in averting human diabetic sensitivity to drug-induced hepatotoxicities. While the rat diabetic models appear to be suitable, the diabetic mouse models might not be suitable in preclinical testing for potential hepatotoxic effects of drugs or toxicants, because regardless of type 1 or type2 diabetes, mice are resistant to acute drug-or toxicant-induced toxicities.

Effects of chronic ethanol on hepatic and renal CYP2C11 in the male rat: interactions with the Janus-kinase 2-signal transducer and activators of transcription proteins 5b pathway.

Chronic alcohol intake in male rats results in: 1) demasculinization of the GH pulse pattern; 2) reduced serum testosterone concentrations; and 3) decreased expression hepatic CYP2C11. Hepatic CYP2C11 expression is regulated by the male pattern of GH through the Janus-kinase/signal transducer and activators of transcription proteins (JAK/STAT) signal transduction pathway in the male rat. Renal CYP2C11 is regulated by testosterone, not GH. The involvement of the JAK/STAT5b signal transduction pathway in renal CYP2C11 signaling has not been studied. We tested the hypothesis that ethanol reduces CYP2C11 levels by interfering with the JAK/STAT5b pathway. Using a total enteral nutrition (TEN) model to feed rats a well-balanced diet, we have studied the effects of chronic ethanol intake (21 d) on hepatic and renal JAK/STAT pathway of adult male rats (8-10/group). We found decreased hepatic and renal expression of CYP2C11 in ethanol-fed rats with concomitant decreases in STAT5b and phospho-STAT5b, decreased in vitro hepatic STAT5b binding to a CYP2C11 promoter element and no effects on hepatic GHR levels. Ethanol caused tissue specific effects in phospho-JAK2 and JAK2, with increased levels in the liver, but decreased JAK2 expression in the kidney. We conclude that ethanol suppression of CYP2C11 expression is clearly associated with reductions in STAT5b levels, but not necessarily in reductions of JAK2 levels. The mechanisms underlying ethanol-induced suppression of STAT5b is yet to be determined, as is the question of whether this is secondary to hormonal effects or a direct ethanol effect.

Soy and whey proteins downregulate DMBA-induced liver and mammary gland CYP1 expression in female rats.

One possible mechanism by which diet may reduce cancer risk is through enhancement of metabolic systems that prevent activation of carcinogens or accelerate carcinogen inactivation. We studied the effects of diet and 7,12-dimethylbenz-(a)anthracene (DMBA) on hepatic and mammary gland CYP1A1, CYP1A2 and CYP1B1 enzymes in female Sprague-Dawley rats. Diets (AIN-93G) were fed from conception to adulthood, and DMBA was given by oral gavage at age 48-50 d. The protein sources of diets were casein (CAS), soy protein isolate (SPI) or whey protein hydrolysate (WPH). The DMBA-induced hepatic ethoxyresorufin-O-deethylase and methoxyresorufin-O-demethylase activities and CYP1A1 protein and mRNA expression were lower (P < 0.05) in SPI-fed rats compared with those fed casein. Differences in mammary gland CYP1 expression were also observed with decreased DMBA induction (P < 0.05) of all three CYP1 proteins and mRNAs in rats fed either SPI or WPH compared with those fed CAS. Most notable were the decreased constitutive and DMBA-induced mammary gland expression of CYP1B1 protein of 93 and 96%, respectively, in the SPI-fed rats relative to the CAS-fed controls. The diet-induced changes in CYP1 enzyme expression were consistent with changes in the AhR and ARNT transcription factors that regulate them. Decreased (P < 0.05) mammary constitutive AhR and ARNT proteins were measured in SPI-fed rats. There was also a 100% increase in constitutive AhR protein in the WPH-fed rats that paralleled a 100% increase in constitutive CYP1B1 protein in the mammary gland. These results demonstrate the importance of diet in regulation of phase I metabolism in liver and mammary gland, and suggest a potential mechanism by which soy or whey proteins reduce DMBA-induced mammary tumor incidence.

Developmental effects and health aspects of soy protein isolate, casein, and whey in male and female rats.

Dietary factors other than the traditional nutrients are found in the so-called functional foods. They are becoming increasingly recognized as potentially important for maintaining good health. Soybeans are rich in such factors thought to help prevent certain chronic diseases. Soy protein isolate (SPI) is one of the three major proteins used in infant formulas sold in the United States, with casein (CAS) and whey (WPH) proteins being the others. We have been studying the health effects of these proteins. Safety concerns have developed over the consumption of soy-based infant formula, partly because of the high circulating levels of the total isoflavones (phytoestrogens) during "critical periods of infant development." There is a paucity of data on developmental, physiological, neurophysiological, behavioral, metabolic, or molecular effects of soy phytochemicals in humans, especially during pregnancy and infancy. We have studied the effects of CAS, SPI, and WPH in short-term, long-term, and multigenerational studies in rats. Aside from minor differences in body weight gain profiles, CAS-, SPI- or WPH-fed rats did not differ in development, organ weights, in vitro hepatic metabolism of testosterone (T), or reproductive performance. However, some endocrine-related functions differed between rats fed these proteins. We found that SPI accelerated puberty in female rats (p < .05) and WPH delayed puberty in males and females, as compared with CAS (p < .05). Gender differences were also found in gonadectomy-induced steroid responses. Male rats had normal serum T levels, but female rats fed SPI had reduced serum 17beta-estradiol concentrations and a blunted 17beta-estradiol response to ovariectomy, as compared to rats fed CAS or WHP (p < .05). Female rats fed SPI or WHP or treated with genistein had reduced incidence of chemically induced mammary cancers (p < .05) compared to CAS controls, with WHP reducing tumor incidence by as much as 50%, findings that replicate previous results from our laboratory. Together, these results suggest gender-specific differences in development and certain endocrine responses among rats fed diets composed of a single protein source such as those used in infant formulas. Whether similar developmental effects occur in human infants is unknown, but unlikely because (1) most infants do not consume such diets throughout life as these rats did, and (2) no such effects have been reported in millions of American infants fed infant formula containing these proteins. The long-term health consequence implications of early diet exposure to SPI and WPH, such as reduced breast cancer incidence, are likely to be very positive.

Skeletal effects of developmental lead exposure in rats.

To identify possible direct and indirect mechanisms underlying the effects of lead on skeletal growth, 3 studies were conducted. In the first study, 1 male and 1 female pup/litter (n = 5 litters), were exposed ad libitum to 0, 825, or 2475 ppm lead acetate in the drinking water from gestational day 4 to euthanasia on day 55. Tibial strength was tested by 3-point bending and plasma levels of vitamin D metabolites were measured. A dose-dependent decrease of the load to failure was demonstrated but only in male pups. No differences in plasma levels of vitamin D metabolites were observed. In the second study, conducted to test if hormone treatment would attenuate the lead deficits, male and female pups were exposed to 0 or 2475 ppm lead acetate and then, from 30-60 days of age, received either saline vehicle, L-dopa, testosterone (males only), dihydrotestosterone (DHT, males only), or estradiol (females only). Lead exposure significantly reduced somatic growth, longitudinal bone growth, and bone strength during the pubertal period. Sex steroid replacement did not restore skeletal parameters in lead-exposed rats. L-Dopa increased plasma insulin-like growth factor 1 (IGF(1)) concentrations, rates of bone growth, and bone strength measures in controls while having no effect in lead-exposed pups. The third study was conducted at 100 days of age, when endocrine parameters have been shown to be normalized, to test for effects of lead exposure on bone formation during tibial limb lengthening (distraction osteogenesis, DO). Both DO gap x-ray density and proximal new endosteal bone formation were decreased in the distraction gaps of the lead-treated animals (p < 0.01). In conclusion, lead exposure reduced somatic growth, longitudinal bone growth, and bone strength during the pubertal period, and these effects could not be reversed by a growth hormone (GH) axis stimulator or by sex-appropriate hormones. Finally, lead exposure appears to specifically inhibit osteoblastogenesis in vivo in adult animals.

Dietary whey protein protects against azoxymethane-induced colon tumors in male rats.

Epidemiological studies have suggested a relationship between diet and colon cancer incidence. Results from animal studies suggest that whey protein, but not casein protein, may provide protective effects against experimentally induced breast cancer in animals. In the current study, we investigated the effects of casein and whey diets on chemically induced colon cancer in male rats. Pregnant female Sprague Dawley rats (days 3-4 of gestation) were maintained on modified AIN-93G diets formulated with a single protein source of either casein or whey. Life-time exposure to these diets was studied in the F1 generation (experiment A) or the F2 generation (experiment B). Male offspring were weaned to the same diets as the dams and were maintained on these diets throughout the study. At age 90 days, all rats received azoxymethane once a week for 2 weeks (s.c., 15 mg/kg). Forty weeks after the last azoxymethane injection, all rats were euthanized, the colon was examined visually for tumors, and each tumor was histologically evaluated. The weights and distribution of all of the tumors were recorded. In experiment A, rats fed the casein diet had a 56% incidence of colon tumors compared with 30% of the rats on whey-based diets (P < 0.05). In experiment B, rats fed the casein diet had 50% incidence of colon tumors compared with 29% in the whey group (P < 0.05). There were no significant effects of diet on tumor multiplicity or mass. These results suggest that consumption of whey protein-containing diets may reduce the risk of developing colon tumors.

Inducibility of hepatic CYP1A enzymes by 3-methylcholanthrene and isosafrole differs in male rats fed diets containing casein, soy protein isolate or whey from conception to adulthood.

Hepatic cytochrome P450 (CYP)1A1 and 1A2 enzymes were studied in male Sprague-Dawley rats derived from 5-7 litters fed diets in which the protein source was casein, soy protein isolate or whey. At age 65 d, rats were gavaged with corn oil (vehicle), 40 mg/kg 3-methylcholanthrene (3-MC) or 75 mg/kg isosafrole (ISO). Hepatic expression of CYP1A1 and CYP1A2 mRNA, apoprotein and associated monooxygenase activities were measured 17 h later. No significant dietary effects were observed on basal expression of either enzyme. However, interactions between diet and the two inducers (3-MC and ISO) were observed in soy-fed rats for ethoxy- and methoxyresorufin O-dealkylase activity, CYP1A1 and CYP1A2 apoprotein and mRNA (P < 0.05). The level of induction of CYP1A1 mRNA and apoprotein was lower in rats fed soy diets than in rats fed casein diets (P < 0.05), and the level of induced CYP1A2 mRNA was lower in rats fed soy or whey (P < 0.05) after treatment with the aryl hydrocarbon (Ah) receptor-dependent inducer 3-MC. This was accompanied by a 50% reduction in constitutive levels of the Ah receptor in liver cytosol of soy-fed, relative to casein-fed rats, and a slightly smaller reduction in whey-fed rats. Expression of the Ah receptor correlated with 3-MC-inducibility of CYP1A1 mRNA in rats fed the three diets. In contrast, in rats induced with ISO, which does not bind to the Ah receptor and induces CYP1As via different mechanisms than 3-MC, ethoxyresorufin O-deethylase activity and levels of CYP1A1 apoprotein and mRNA were elevated to a greater degree in soy-fed than in casein- or whey-fed rats (P < 0.05). Moreover, after ISO treatment, induction of methoxyresorufin O-demethylase activity, CYP1A2 apoprotein and mRNA levels was observed only in rats fed soy (P < 0.05). These data suggest potential effects of dietary protein source on metabolism of a wide variety of CYP1A substrates, including environmental and dietary carcinogens, many of which induce their own metabolism.

Soy protein isolate consumption protects against azoxymethane-induced colon tumors in male rats.

Male Sprague-Dawley rats (F2 generation) that had been fed modified American Institute of Nutrition-93G diets formulated with a single protein source of either casein or soy protein isolate for their entire life received azoxymethane once a week for 2 weeks (s.c., 15 mg/kg) starting at age 90 days. Forty weeks later, all rats were euthanized, the colon was examined visually for masses and these were subsequently evaluated histologically. Rats fed the casein diet had a 50% incidence of colon tumors compared with 12% on soy protein-based diets (P<0.05). These results suggest that consumption of soy protein-containing diets may reduce the risk of developing colon tumors.

Chronic intragastric infusion of ethanol-containing diets induces CYP3A9 while decreasing CYP3A2 in male rats.

The CYP3A subfamily is the most abundant of the human hepatic cytochrome P450 enzymes. They mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. We studied diet/ethanol interactions using total enteral nutrition in adult male Sprague-Dawley rats with diets containing 16% protein, ethanol (13 g/kg), corn oil (fat; 25-45%), and carbohydrate (CHO; 1-21%). Using this model, chronic ethanol feeding decreased CYP3A activity (testosterone 6 beta-hydroxylation) and apoprotein levels (Western blot) (P <.05) and these effects were independent of the dietary CHO/fat ratio. The CYP3A2 mRNA levels decreased (P <.05) in the rats fed ethanol-containing diets by 73 to 83% compared with rats fed control diets, regardless of the CHO/fat ratio. In contrast, ethanol induced CYP3A9 mRNA levels (P <.05) and this effect was greater (P <.05) in the high-CHO/low-fat group (11.3-fold) than in the low-CHO/high-fat group (2.6-fold). Purified recombinant rat P450 3A9 had a chlorzoxazone 6-hydroxylase activity with a turnover number 1.3 nmol/min/nmol of P450. These results indicate that 1) ethanol differentially affects the expression of CYP3A gene family and this regulation appears to be modulated by dietary CHO/fat ratio; 2) the decrease in testosterone 6 beta-hydroxylase activity and CYP3A apoprotein levels are most likely due to the ethanol-induced decrease in CYP3A2 mRNA levels; and 3) CYP3A9 is induced by ethanol and is a low-affinity, high-K(m) chlorzoxazone hydroxylase.

Cyclic expression of class I alcohol dehydrogenase in male rats treated with ethanol.

Continuous infusion of ethanol-containing diets has been demonstrated to generate well-defined pulses in blood and urine ethanol concentrations that occur with a frequency of approximately 6 days. The present study aimed to determine if hepatic class I alcohol dehydrogenase was the cause of these cycles. Adult male rats were fed an ethanol-containing diet by continuous intragastric infusion. Hepatic ADH activity, class I ADH mRNA level and rate of class I ADH gene transcription fluctuated in a cyclic pattern that positively correlated with UECs, and inhibition of ADH with 4-methylpyrazole abolished the UEC pulses. These data demonstrate for the first time an ethanol-dependent regulation of rat hepatic class I ADH. The cyclic behavior of the ethanol levels correlates with changes in class I ADH expression and implies adaptability of the ethanol eliminating system to high concentrations of alcohol.

Potentiation of thioacetamide liver injury in diabetic rats is due to induced CYP2E1.

Thioacetamide (TA)-induced hepatotoxicity is potentiated in streptozotocin (STZ)-induced diabetic rats. The relative roles of CYP2E1 and FMO1 in the mechanism of TA-associated liver injury were investigated. In the STZ-induced diabetic rat, hepatic CYP2E1 protein concentration and p-nitrophenol hydroxylation were induced 8- and 5.6-fold, respectively. Pretreatment with the CYP2E1 inducer, isoniazid (INH, 250 mg/kg, i.p.) before TA (300 mg/kg, i.p.) administration significantly increased TA-associated liver injury as assessed by plasma alanine aminotransferase (ALT). Hepatic CYP2E1 expression and p-nitrophenol hydroxylation were induced 2.2- and 2. 5-fold in the INH-pretreated rat, respectively. Inhibition of CYP2E1 by diallyl sulfide (DAS, 200 mg/kg, p.o., two doses) before TA administration, decreased plasma ALT activity by 60% in the nondiabetic rat and by 75% in the diabetic rat. Abolition of microsomal p-nitrophenol hydroxylation and CCl(4)-induced liver injury confirmed that hepatic CYP2E1 was highly inhibited by DAS. Hepatic flavin-containing monooxygenase (FMO) form 1 expression and methimazole-dependent oxidation of thiocholine were induced 2.5- and 1.8-fold in the diabetic rat, respectively. Dietary administration of 0.25% indole-3-carbinol (I3C) for 10 days inhibited FMO1 expression and enzyme activity in both nondiabetic and diabetic rats. Paradoxically, TA-induced liver injury was increased in these I3C-pretreated rats. These findings indicate that hepatic CYP2E1 appears to be primarily involved in bioactivation of TA. In the STZ-induced diabetic rat, diabetes-induced CYP2E1 appears to be responsible for the potentiated liver injury; Even though hepatic FMO1 is induced in the diabetic rat, it is unlikely to mediate the potentiated TA hepatotoxicity.

Diets containing whey proteins or soy protein isolate protect against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in female rats.

A study was conducted to determine the protective effects of two common dietary proteins, soy protein isolate (soy) and bovine whey, against chemically induced mammary tumors in female Sprague Dawley rats. Rats were fed AIN-93G diets having casein, soy, or whey as the sole protein source. Rats within the same dietary groups were mated to obtain the F1 and F2 generations. At age 50 days, F1 (experiment A) or F2 (experiment B) female offspring (> or =19 rats/group) were p.o. gavaged with 80 mg/kg 7,12-dimethylbenz(a)anthracene, and mammary glands were evaluated when 100% of the casein-fed group developed at least one palpable tumor. Rats grew well on all three diets, but casein-fed rats gained slightly more body weight than soy- or whey-fed rats (P < 0.05). Vaginal opening occurred 1 day earlier in soy-fed rats than in casein- or whey-fed rats, but no other differences in reproductive and developmental parameters were observed between groups. When 50% of the casein-fed rats had at least one mammary tumor, lower tumor incidences (24-34%) were observed in the soy-fed (P < 0.009) and whey-fed groups (P < 0.001). When 100% of the casein-fed rats had at least one tumor, soy-fed rats had a lower tumor incidence (77%) in experiment B (P < 0.002), but not in experiment A (P < 0.12), and there were no differences in tumor multiplicity. Whey-fed rats had lower mammary tumor incidence (54-62%; P < 0.002) and multiplicity (P < 0.007) than casein-fed rats in both experiments. Our results indicate that diets rich in soy reduce the incidence of chemically induced mammary tumors by approximately 20%. Furthermore, whey appears to be at least twice as effective as soy in reducing both tumor incidence and multiplicity.

An LC-MS method to determine concentrations of isoflavones and their sulfate and glucuronide conjugates in urine.

Most methods for detecting isoflavones in biological samples do not measure the concentration of sulfate conjugates. An LC-MS method is reported here to estimate urinary concentrations of genistein and daidzein, their sulfate and glucuronide conjugates and other major metabolites. Human and rat urine samples were extracted with diethyl ether, or pre-digested with sulfatase and/or beta-glucuronidase followed by extraction. The isoflavones were separated using gradient LC methods and detected by negative single ion monitoring on an MS system using a heated nebulizer atmospheric pressure chemical ionization interface. CVs for inter- and intra-assay variability were generally < 20 and 10%, respectively. Preliminary studies using these procedures demonstrate 52+/-4 and 26+/-4% of genistein in rat urine was found as the aglycone and sulfate conjugates, respectively, compared to 0.36 and 4%, respectively, in human urine. This method is suitable for the study of isoflavone sulfate conjugates in biological fluids.

Altered expression and glucocorticoid-inducibility of hepatic CYP3A and CYP2B enzymes in male rats fed diets containing soy protein isolate.

Hepatic CYP3A and CYP2B enzymes were studied in male Sprague-Dawley rats derived from 5-7 litters fed diets in which the protein source was either casein or soy protein isolate. At age 65 d, rats were gavaged with corn oil (vehicle) or 50 mg/kg dexamethasone. Hepatic expression of CYP3A and CYP2B1 mRNA, apoprotein and associated monooxygenase activities were measured. Consumption of soy diets significantly increased monooxygenase activity toward the following: the CYP3A substrates erythromycin and ethylmorphine N-demethylase; corticosterone and testosterone 6beta-hydroxylase; and apoprotein and mRNA expression of CYP3A2 (P < 0.05). Dexamethasone significantly induced turnover of erythromycin and testosterone, expression of CYP3A apoprotein, and expression of CYP3A1 and CYP3A2 mRNA (P < 0.05). In addition, significant diet-inducer interactions were observed in the expression of CYP3A apoprotein and activities toward ethylmorphine, corticosterone and testosterone (P < 0.05). Significant diet-inducer interactions were also observed on CYP2B1-dependent pentoxyresorufin O-depentylase activity (P < 0.05). However, although dexamethasone significantly induced CYP2B1 expression at the apoprotein and mRNA level (P < 0.05), no significant diet effects were observed. These data suggest potential effects of soy consumption on the metabolism of a wide variety of CYP3A and CYP2B1 substrates, especially in situations involving coexposure to CYP inducers.

Hormonal regulation of microsomal cytochrome P4502E1 and P450 reductase in rat liver and kidney.

1. The relative roles of pituitary hormones (especially growth hormone) and testicular hormones (especially testosterone) in the regulation of renal and hepatic CYP2E1 and cytochrome P450 reductase have been studied in the male rat. 2. Depletion of pituitary hormones by hypophysectomy (Hx) resulted in 12-14-fold increases in renal CYP2E1 (p < or = 0.05) and a 40% drop in NADPH-dependent cytochrome c reductase activity (p < or = 0.05) compared with 6-fold increases in CYP2E1 (p < or = 0.05) and a 60% drop in P450 reductase apoprotein (p < or = 0.05) in the liver. 3. The increase in hepatic CYP2E1 was associated with increased gene transcription in nuclear run-on experiments. 4. Restoration of renal CYP2E1 to control levels by hormone treatment required both growth hormone and an intact testis, whereas partial restoration of CYP2E1 apoprotein levels in liver was accomplished by growth hormone, but not testosterone. 5. Renal NADPH-dependent cytochrome c reductase activity was restored by growth hormone and testosterone treatment, whereas the hepatic reductase appeared to be regulated by other pituitary hormones. 6. CYP2E1 and P450 reductase appear to be under complex endocrine regulation by pituitary and testicular hormones in a tissue specific manner.