Livers Of Female Sprague-Dawley Rats Research Articles

DNA Product Formation in Female Sprague-Dawley Rats Following Polyhalogenated Aromatic Hydrocarbon (PHAH) Exposure.

DNA oxidation damage has been regarded as one of the possible mechanisms for the hepatic carcinogenesis of dioxin-like compounds (DLCs). In this study, we evaluated the toxic equivalency factor (TEF) from the standpoint of induced DNA oxidation products and their relationship to toxicity and carcinogenicity. Nine DNA oxidation products were analyzed in the liver of female Sprague–Dawley rats exposed to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) alone or the tertiary mixture of TCDD, 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126), and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) by gavage for 14, 31, and 53 weeks (5 days/week) by LC–MS/MS: 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dGuo); 1,N6-etheno-2′-deoxyadenosine (1,N6-εdAdo); N2,3-ethenoguanine (N2,3-εG); 7-(2-oxoethly)guanine (7-OEG); 1,N2-etheno-2′-deoxyguanosine (1,N2-εdGuo); malondialdehyde (M1dGuo); acrolein (AcrdGuo); crotonaldehyde (CrdGuo); and 4-hydroxynonenal (HNEdGuo) derived 2′-deoxyguanosine adducts. Exposure to TCDD (100 ng/kg/day) significantly induced 1,N6-εdAdo at 31 and 53 weeks, while no increase of 8-oxo-dGuo was observed. Significant increases were observed for 8-oxo-dGuo and 1,N6-εdAdo at all time points following exposure to the tertiary mixture (TEQ 100 ng/kg/day). Exposure to TCDD for 53 weeks only significantly increased 1,N6-εdAdo, while increases of N2,3-εG and 7-OEG were only found in the highest dose group (100 ng/kg/day). Exposure to the tertiary mixture for 53 weeks had no effect on N2,3-εG in any exposure group (TEQ 0, 22, 46, or 100 ng/kg/day), while significant increases were observed for 1,N6-εdAdo (all dose groups), 8-oxo-dGuo (46 and 100 ng/kg/day), and 7-OEG (100 ng/kg/day). While no significant increase was observed at 53 weeks for 1,N2-εdGuo, M1dGuo, AcrdGuo, or CrdGuo following exposure to TCDD (100 ng/kg/day), all of them were significantly induced in animals exposed to the tertiary mixture (TEQ 100 ng/kg/day). This oxidation DNA product data suggest that the simple TEF methodology cannot be applied to evaluate the diverse patterns of toxic effects induced by DLCs.

Common Handling Procedures Conducted in Preclinical Safety Studies Result in Minimal Hepatic Gene Expression Changes in Sprague-Dawley Rats

Gene expression profiling is a tool to gain mechanistic understanding of adverse effects in response to compound exposure. However, little is known about how the common handling procedures of experimental animals during a preclinical study alter baseline gene expression. We report gene expression changes in the livers of female Sprague-Dawley rats following common handling procedures. Baseline gene expression changes identified in this study provide insight on how these changes may affect interpretation of gene expression profiles following compound exposure. Rats were divided into three groups. One group was not subjected to handling procedures and served as controls for both handled groups. Animals in the other two groups were weighed, subjected to restraint in Broome restrainers, and administered water via oral gavage daily for 1 or 4 days with tail vein blood collections at 1, 2, 4, and 8 hours postdose on days 1 and 4. Significantly altered genes were identified in livers of animals following 1 or 4 days of handling when compared to the unhandled animals. Gene changes in animals handled for 4 days were similar to those handled for 1 day, suggesting a lack of habituation. The altered genes were primarily immune function related genes. These findings, along with a correlating increase in corticosterone levels suggest that common handling procedures may cause a minor immune system perturbance.

The Disposition of Morphine and Morphine-3-glucuronide in the Isolated Perfused Rat Liver: Effects of Altered Perfusate Flow Rate

The rat single-pass isolated perfused liver preparation was used to study the effects of altered perfusate flow rate on the hepatic disposition of morphine and its polar metabolite morphine-3-glucuronide (M3G). Using a balanced, cross-over design, livers of female Sprague-Dawley rats (n = 6) were perfused at 15 and 30 mL min-1 with erythrocyte- and protein-free perfusion medium containing a constant concentration of morphine (2.7 microM). After reaching steady-state, inflow and outflow perfusate and bile samples were collected and morphine and M3G were measured by HPLC. Doubling of perfusate flow rate was associated with a significant increase (P < 0.05) in the availability of morphine (mean +/- s.d. of 0.19 +/- 0.06 at 15 mL min-1 and 0.29 +/- 0.08 at 30 mL min-1). The magnitude of the change in morphine availability was consistent with the predictions of the well-stirred model of hepatic elimination. The fate of hepatically generated M3G was assessed by the biliary extraction ratio of M3G; alterations in perfusate flow rate had no significant effect on this ratio (mean +/- s.d. of 0.49 +/- 0.14 at a perfusate flow rate of 15 mL min-1 and 0.47 +/- 0.22 at 30 mL min-1). A physiologically-based mathematical model, in which the vascular and intracellular spaces of the liver were represented by two well-mixed compartments, was utilized to derive an equation for the biliary extraction ratio of M3G. According to the model, the value of this extraction ratio will become insensitive to changes in perfusate flow rate when the permeability for M3G of the membrane separating the intracellular and vascular compartments is low compared with perfusate flow rate. Hence, the experimental results are consistent with the concept that the hepatic sinusoidal membrane represents a diffusional barrier to M3G.

Liver AMP/ATP ratio and fructokinase expression are related to gender differences in AMPK activity and glucose intolerance in rats ingesting liquid fructose

Women, but not men, show an association between fructose consumption and an increased risk of Type 2 diabetes mellitus. As rats are considered a model for human fructose metabolism, we sought to determine whether such a gender-related difference is present in Sprague-Dawley rats and to analyze the molecular mechanism behind. Male and female Sprague-Dawley rats had free access to water or to a 10% w/v fructose solution for 14 days. Plasma analytes, liver triglycerides and enzyme activities and the expression of enzymes and transcription factors related to fatty acid metabolism, insulin signaling and glucose tolerance were determined. Fructose-fed rats had hypertriglyceridemia, steatosis and reduced fatty acid oxidation activity, although the metabolic pattern of fructose-fed female rats was different to that observed for male rats. Fructose-fed female, but not male rats, showed no change in plasma leptin; they had hyperinsulinemia, an altered glucose tolerance test and less liver insulin receptor substrate-2. Further, only fructose-fed female rats had increased adenosine 5'-monophosphate (AMP)-activated protein kinase activity, resulting in a decreased expression of hepatic nuclear factor 4 and sterol response element binding protein 1. These differences were related to the fact that liver expression of the enzyme fructokinase, controlling fructose metabolism, was markedly induced by fructose ingestion in female, but not in male rats, resulting in a significant increase in the AMP/adenosine 5'-triphosphate (ATP) ratio and, thus, AMP-activated protein kinase activation, in female rats only. The difference in fructokinase induction could explain the higher metabolic burden produced by fructose ingestion in the livers of female Sprague-Dawley rats.

All‐trans Retinoic Acid Induction of Sulfotransferases

All-trans retinoic acid is the bioactive form of vitamin A (retinol). Retinoids have been used clinically as therapeutic agents against a number of cancers. Retinoids have been reported to induce the phase I drug metabolizing enzymes, cytochrome P-450s. In contrast, effects of retinoids on sulfotransferases have not been as well studied. The present investigation evaluates the role of retinoic acid on the expression of aryl sulfotransferase IV and hydroxysteroid sulfotransferase a in male and female Sprague-Dawley rat liver and intestine. Cultured human hepatic carcinoma cells (Hep G2) and intestinal carcinoma cells (Caco-2) were also used to study retinoic acid's effect on simple phenol sulfating sulfotransferase, dehydroepiandrosterone sulfotransferase and oestrogen sulfotransferase. Enzyme assay and Western blot were used to determine sulfotransferase protein expression. Retinoic acid induced aryl sulfotransferase IV in liver of female rats and sulfotransferase a in liver of male rats. Intestinal rat aryl sulfotransferase IV and sulfotransferase a in male rats and intestinal aryl sulfotransferase IV in female rats were also induced after retinoic acid treatment. In Hep G2 and Caco-2 cells, retinoic acid differentially induced the three human sulfotransferase isoforms. In general, intestinal sulfotransferases were found to be more responsive than hepatic sulfotransferases to retinoic acid treatment. mRNA expressions were investigated using reverse transcription polymerase chain reaction with gene specific primers. Reverse transcription polymerase chain reaction results are in good agreement with enzyme activity and Western blot results. This suggests that retinoic acid induction of sulfotransferases is at the transcriptional level.

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 μg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 μg TCDD/kg bw. Only 4.0 μg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 μg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-α, IL-1β and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.

Characterization of the major DNA adduct formed by alpha-hydroxy-N-desmethyltamoxifen in vitro and in vivo.

Tamoxifen is hepatocarcinogenic in rats and has been associated with an increased risk of endometrial cancer in women. Recent reports suggest that it may be genotoxic in humans. N-Desmethyltamoxifen is a major tamoxifen metabolite that has been proposed to be responsible for one of the major adducts detected in liver DNA of rats treated with tamoxifen. The metabolic activation of N-desmethyltamoxifen to DNA binding products may involve oxidation to alpha-hydroxy-N-desmethyltamoxifen followed by esterification. In the study presented here, we report the synthesis of alpha-hydroxy-N-desmethyltamoxifen and the characterization of the major adduct obtained from alpha-sulfoxy-N-desmethyltamoxifen in vitro as (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen. In addition, we use (32)P-postlabeling in combination with HPLC to compare the adducts formed in the livers of female Sprague-Dawley rats treated by gavage with tamoxifen or equimolar doses of alpha-hydroxy-N-desmethyltamoxifen. We conclude that one of the major adducts formed in vivo and previously suggested to derive from N-desmethyltamoxifen is chromatographically identical to alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen.

Differences in kinetics of induction and reversibility of TCDD-induced changes in cell proliferation and CYP1A1 expression in female Sprague-Dawley rat liver.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent tumor promoter in two-stage initiation-promotion models and induces cell proliferation and development of enzyme-altered hepatic foci. It is believed that increased cell proliferation is a necessary step in carcinogenesis. Therefore, the analysis of the effect of TCDD on cell proliferation in rat liver may aid in the understanding of the mechanism of hepatocarcinogenesis induced by TCDD. The aim of this study was to investigate the time course and reversibility of cell proliferation in non-initiated and diethylnitrosamine-initiated female rats exposed biweekly to a daily averaged dose of 125 ng TCDD/kg/day for up to 60 weeks. In addition we evaluated the suitability of different dose metrics for the evaluation of TCDD-induced changes in cell proliferation and CYP1A1 enzyme induction. Cell proliferation was measured as the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into hepatocytes undergoing replicative DNA synthesis. Mean BrdU labeling indices in TCDD-treated animals were not increased over controls after 14 weeks exposure, but were increased 8- and 2-fold after 30 and 60 weeks' treatment respectively, despite similar liver levels of TCDD at all these times (23-30 p.p.b.). In comparison, CYP1A1 activity, as measured by ethoxyresorufin deethylase activity, was significantly induced at all times points analyzed. Sixteen weeks following cessation of TCDD treatment, labeling indices were still significantly elevated over controls, but after 30 weeks of withdrawal, labeling indices were no different from controls, indicating that TCDD-induced changes in cell proliferation were reversible. Dosimetric analysis indicated that rat liver tissue burden was suitable for prediction of CYP1A1 expression but not cell proliferation and that the area under the curve was unsuitable for prediction of both TCDD-induced changes in CYP1A1 expression and cell proliferation.

DNA adduct formation by tamoxifen and structurally-related compounds in rat liver

Binding of diethylstilbestrol and four different triphenylethylene derivatives: tamoxifen, toremifene, clomiphene and triparanol to DNA in rat liver, was studied using the 32P-postlabelling method with HPLC-radioactivity detection. Three different modifications of the 32P-postlabelling technique (a) a bisphosphate method with adduct enrichment by nuclease P1 (NP1)-treatment or (b) by butanol extraction and (c) a monophosphate method, were applied in order to provide an unbiased analysis of adduct formation. When tamoxifen was administered by daily gavage for 4 weeks (80 μmol/kg for 2 weeks and 40 μmol/kg for a further 2 weeks) two major adducts and about six minor adducts were produced in the liver of female Sprague-Dawley rats. Equimolar doses of toremifene produced one apparent adduct. The adduct levels in the tamoxifen and toremifene treated rats were 600 and 2/10 8 nucleotides, respectively. Under conditions used, clomiphene, triparanol and diethylstilbestrol did not produce DNA adducts. The present and previous data suggest that modification (a) is the 32P-postlabelling method of choice for risk assessment in human subjects. Modification (c) with butanol extraction after labelling has the advantage of low background radioactivity and may be preferable if large amounts of DNA are available. The main tamoxifen adducts were suggested to be α-( N 2-deoxyguanosinyl)tamoxifen and α-( N 2-deoxyguanosinyl)- N-desmethyltamoxifen.

Induction and localization of cytochrome P450 1B1 (CYP1B1) protein in the livers of TCDD-treated rats: detection using polyclonal antibodies raised to histidine-tagged fusion proteins produced and purified from bacteria.

Knowledge of the response of cytochrome P450 1B1 (CYP1B1) to exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in both humans and rodents is limited. To improve the analysis of CYP1 proteins, specific CYP1B1 and CYP1A1 polypeptides were expressed as hexahistidine-tagged fusion proteins in Escherichia coli, purified to homogeneity and used to produce polyclonal antibodies in rabbits. Immunoblot analyses showed that these antibodies were specific and sensitive, detecting both the human and rat forms of the respective isozymes and exhibiting negligible cross-reactivity between the two known CYP1 subfamilies. We show that CYP1B1, CYP1A1 and CYP1A2 protein levels were induced in the livers of female Sprague-Dawley rats following either acute (single dose of 25 microg TCDD/kg) or chronic (125 ng TCDD/kg/day for 30 weeks) exposure to TCDD. CYP1B1 protein exhibited a dose-response to TCDD that was different from those of CYP1A1 and CYP1A2. CYP1B1 induction appeared to be less sensitive to TCDD exposure, with induction occurring at higher doses of TCDD than that required for induction of CYP1A1 or CYP1A2. Immunohistochemical analysis showed that in animals chronically exposed to TCDD (35 ng/kg/day for 30 weeks), CYP1B1 was induced only in centrilobular hepatocytes, a pattern of expression similar to that of CYP1A1 and CYP1A2. These observations of cellular co-localization of the CYP1 cytochromes in livers of TCDD-treated rats and apparent differences in both protein amounts and dose-response are indicative of both common and unique regulation of CYP1 induction.

The ability to alter the gap junction protein expression outside GST-P positive foci in liver of rats was associated to the tumour promotion potency of different polychlorinated biphenyls

The results demonstrate different modes of action by a dioxin-like polychlorinated biphenyl (PCB 126) and a non dioxin-like PCB (PCB 153) in the alteration of connexin (cx) 26 and cx 32 expression outside GST-P positive foci in liver of female Sprague-Dawley rats, treated according to an initiation-promotion protocol. A decreased relative amount of immunopositive cx 26 and cx 32 spots in the parenchymal cell plasma membranes was observed after treatment with the potent tumour promoters PCB 126 or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). No reduction of cx 26 or cx 32 was noted after administration with the weaker tumour promoters PCB 153 or PCB 118 (PCB 118; both dioxin- and non dioxin-like). Additionally, we found that the down-regulation of connexins also occurred in rats treated with PCB 126 or TCDD without partial hepatectomy and initiation with nitrosodiethylamine. In summary, the results indicate that the ability to reduce the gap junction protein level in liver of rats can be associated to the tumour promotive potency of the different PCB-congeners and TCDD.

Debenzoylating and deacetylating activities of rat liver and mammary gland microsomes: Effect of ovariectomy

This study compared the rates of N-deacylations of N-hydroxy- N-2-fluorenylbenzamide (N-OH-2-FBA) with those of its analogue, N-hydroxy- N-2-fluorenylacetamide (N-OH-2-FAA), by the mammary gland (tumor target for both compounds) and the liver of female Sprague-Dawley rats and examined the effect of ovariectomy on these activities. N-Debenzoylation of N-OH-2-FBA was catalyzed by the mammary and liver microsomes of 50-day-old female rats at similar rates (~24 nmol/min/mg). The activity of both tissues increased (up to 1.8 times) after ovariectomy at 42, 32 and, especially, 22 days of age. The rapid hydrolysis appeared to be unique for the benzoyl group since N-OH-2-FAA was deacylated only ~0.05 and 0.004 times as fast by the liver and mammary microsomes, respectively, and these low rates were unaffected by ovariectomy. Since such substrate specificity would be of significance in the metabolism of xenobiotics and drug design, esterase activity and its sensitivity to ovariectomy at 22 days of age were examined with several acetylated and benzoylated substrates in the liver and mammary microsomes and compared with those of male liver. Tissues of rats of both sexes had a greater capacity to hydrolyze carboxyl esters than amides. Except for N-2-fluorenylacetamide (2-FAA) and o-nitrophenylacetate (o-NPA), all substrates were hydrolyzed by liver microsomes of the male up to 3.9 times faster than by those of the female. Microsomes of female liver hydrolyzed acetylated substrates 1.2 to 25 times faster than benzoylated analogues except for N-OH-2-FBA and benzamide. By contrast, mammary gland microsomes hydrolyzed benzoylated compounds 1.4 to 333 times faster except for 2-naphthyl benzoate. Respective rates of hydrolysis of o-NPA by microsomes of liver and mammary gland were 1.7 and 0.6 times those of p-NPA. After ovariectomy, deacylating activities increased (up to 1.6 times) except for those of 2-FAA and acetanilide. All deacylations were > 98% inhibited by 0.1 mM paraoxon, indicating catalysis by serine hydrolases. The results suggest involvement of multiple carboxylesterases and indicate that certain benzoylated xenobiotics may have a greater effect on the mammary gland than acetylated xenobiotics because of their greater vulnerability to hydrolysis by esterases of mammary gland.

Microsomal and peroxidase activation of 4-hydroxy-tamoxifen to form DNA adducts: comparison with DNA adducts formed in Sprague-Dawley rats treated with tamoxifen

Using rat liver microsomal preparations and peroxidase enzymes, we have investigated the formation of DNA adducts by the antiestrogen compound tamoxifen (TAM) and its metabolite 4-hydroxy-tamoxifen (4-OH-TAM). When reduced nicotinamide-adenine dinucleotide phosphate (NADPH) was used as a cofactor in microsomal activation of either 4-OH-TAM or TAM, one DNA adduct and relative DNA adduct levels of 4.6 and 3.1 x 10(-8), respectively were detected by 32P-postlabeling. The DNA adduct produced by microsomal activation of 4-OH-TAM and TAM was the same. With cumene hydroperoxide (CuOOH) as the cofactor for the microsomal activation of either 4-OH-TAM or TAM, three to six DNA adducts were produced; the relative adduct levels were 8.0 and 20.6 x 10(-8), respectively. Comparison of the DNA adduct patterns produced by 4-OH-TAM and TAM showed that they were distinct. However one of the DNA adducts (a) produced by microsomal activation of 4-OH-TAM using CuOOH was the same as adduct a produced by microsomal activation of 4-OH-TAM with NADPH. Activation of 4-OH-TAM with horseradish peroxidase resulted in the formation of a single DNA adduct and a relative adduct level of 20.7 x 10(-8). Rechromatography analysis of this DNA adduct showed that it was identical to that produced by microsomal activation of 4-OH-TAM with NADPH and one of the adducts produced using CuOOH as the cofactor. Ten DNA adducts and a relative adduct level of 15.3 x 10(-8) were detected in the liver of female Sprague-Dawley rats treated daily with 20 mg/kg of TAM for 7 days. The DNA adduct pattern in the liver of the treated animals was similar to that produced by microsomal activation of TAM using CuOOH as the co-factor. The principal DNA adduct (no. 6) formed in the livers of rats treated with TAM was the same as the principal DNA adduct formed following microsomal activation of TAM using CuOOH as a cofactor. The DNA adduct formed following microsomal activation of either TAM or 4-OH-TAM using NADPH was also present as one of the adducts (1) formed in vivo following TAM treatment. These studies demonstrate that 4-OH-TAM can be activated to form DNA adducts and that it contributes to the formation of DNA adducts in the liver of rats treated with TAM.

Major hydroxysteroid sulfotransferase STa in rat liver cytosol may consist of two microheterogeneous subunits

The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries. These subunits consisted of an equal number of amino acid residues with only one amino acid substitution. ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol. This strongly suggested that they are essential components of STa. The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.

Tamoxifen induces hepatocellular carcinoma in rat liver: A 1-year study with two antiestrogens

The effects of equimolar doses of the triphenylethylene antiestrogens tamoxifen and toremifene on female Sprague-Dawley rat liver were studied in a 52-week toxicity study which included a 13-week recovery period. Liver tumors were found in four out of five rats at the highest dose level of tamoxifen (45 mg/kg per day) after 52 weeks of dosing, and these appeared to be hepatocellular carcinomas in three rats. After the 13-week recovery period all surviving rats in the highest tamoxifen dose group had large liver tumors (diameter up to 2 cm) which appeared to be hepatocellular carcinomas in five out of six rats. No tumor was observed in the toremifene-treated rats (48 mg/kg per day) either after 52 weeks of dosing or after the recovery period. Electron microscopic morphometric analysis after 52 weeks of dosing revealed that at the tamoxifen high dose level, the volume densities of the peroxisomes, mitochondria, and residual bodies were elevated in the nonneoplastic hepatocytes of the rats. In the neoplastic hepatocytes of the tamoxifen-treated rats the volume density of nuclei was slightly elevated. The slight proliferation of peroxisomes and mitochondria might be related to tumor development in the tamoxifen treated rats.

Rat Liver Hydroxysteroid Sulfotransferases That Are Very Difficult to Separate: an Approach by Molecular Cloning

Possible existence of five different subunits, designated ST-20P, ST-21P, ST-40P, ST-41P, and ST-60P, of hydroxysteroid (H) sulfotransferases (ST's) in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNA's isolated from both of two rat liver cDNA libraries. The HST subunits consisted of 284 amino acid residues (Mr 33, 084-33, 256) except ST-60P (Mr 33, 535) which had an additional Met as the second amino acid residue. They shared extremely strong or very strong homology (>83%) in amino acid sequence with one another, . The HST subunits, however, shared very weak (<40%) homology with those of rat liver phenol and estrogen ST's. Of the three rat liver cytosolic HST's, STa, STb, and STc, which were separable on an anion-exchange column by direct application of the cytosol and all catalyzed sulfation of dehydroepiandrosterone and bioactivation by sulfation of the potent carcinogen, 5-hydroxymethylchrysene (5-HCR), the major enzyme STa was strongly suggested by the study on expression of cDNA's encoding ST-40P and ST-41P in Escherichia coli to consist of these microheterogeneous subunits with only one amino acid substitution. The recombinant proteins showed enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and 5-HCR at rates very similar to those by STa in the presence of Tween 20. The detergent that was found to be a powerful HST stabilizer completely prevented the hepatic and recombinant HST's from facile aggregation with marked loss of their enzyme activities. In the presence of Tween 20, the recombinant HST's and STa existed as dimers and were chromatographically and electrophoretically identical. STa, STb, and STc had an equal size of subunits (30.5 kDa) which were all immunostained with anti-STa-antibody, although HST's in the rat liver cytosol had been reported to have different sizes of subunits (28-60 kDa)without immuno-homology. The present study carried out using the recombinant enzymes provides the first direct evidence for the identity of ST's catalyzing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing HST's from bile acid STs should be improved.

Oat lipids-induced covalent DNA modifications (I-compounds) in female Sprague-Dawley rats, as determined by 32P-postlabeling

Previous studies have shown that the presence of oats in the diet contributes to formation of I-compounds (age-dependent covalent DNA modifications detected by 32P-postlabeling assay) in female Sprague-Dawley rat liver DNA. The current study explored the possible ingredients in oats responsible for the observed effects on DNA. Feeding AIN-76A diet containing 5% oat lipids (obtained by methanol extraction and dissolved in trioctanoin) in place of corn oil for 2 months successfully induced the formation of 3 oats-specific (spots 2–4) and 4 natural ingredient diet-specific I-compounds (spots 6–9) in liver DNA. Barley, an oatlike cereal, induced 3 of these spots at very low intensities but not the 3 oats-specific I-spots. Oral administration of oat lipids to weanling rats of both sexes for 7 days elicited trace amounts of the oats-specific spots and spot 9 in liver DNA. However, when oat lipids were given at 6 or 9 weeks of age, the oats-specific spots were detected at high levels in female but not in male rats. These oats-related DNA modifications were also present in 6-week-old female rats which had received oat lipids p.o. for 2 or 3 days or i.p. for 4 days. Rats given trioctanoin or extracts from natural ingredient Wayne diet (lacking oats) did not show any of these spots. On the other hand, rats treated with extracts from an oats-containing Teklad diet displayed a trace amount of one of these I-compounds. Oat lipids did not induce any extra spots in rat kidney DNA. Feeding of AIN diet supplemented with oats to female Syrian hamsters did not elicit any renal or hepatic DNA alterations, as detected by 32P-postlabeling. Rats fed oat lipids-supplemented AIN diet or Purina diet showed the highest levels of I-compounds overall in liver among all dietary groups and these two groups also had significantly higher hepatic DNA synthesis rates. Oat lipids enhanced kidney DNA synthesis also. The total hepatic or renal cytochrome P-450 contents were not significantly affected by different diets. These results demonstrate a novel link between a natural dietary ingredient and covalent DNA modifications and shed light on the origins of certain I-compounds.

Structure-dependent reduction in DNA I-compound adducts by polychlorinated dibenzofurans in female Sprague-Dawley rat liver

The effects of polychlorinated dibenzofurans (PCDFs) on hepatic I-compound DNA adducts in female Sprague-Dawley rats were determined at 2 dose levels. In the first experiments, rats (4 per group) were treated with each of 3 polychlorinated dibenzofuran (PCDF) congeners at 20 μg/kg/week or 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) at 10 μg/kg/week for 4 weeks. Effects on total I-compound levels when compared with age-matched control animals (corn oil) were as follows: control (116) > 1,2,4,7,8-pentachlorodibenzofuran (PeCDF) (112) > 1,2,3,7,8-PeCDF (93) > 2,3,4,7,8-PeCDF (91) > TCDD (72) where the numbers in parentheses were the number of I-compounds in 10 9 DNA nucleotides. In the second experiment, rats (5 per group) were treated with each of the congeners at a higher dose of 100 μg/kg/week for 4 weeks. The effects were as follows: control (123) ≈ 1,2,4,7,8-PeCDF (125) > 1,2,3,7,8-PeCDF (98) > 2,3,4,7,8-PeCDF (75) ≈ 2,3,4,6,7,8-hexachlorodibenzofuran (HCDF) (75). The active compounds (1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF and 2,3,4,6,7,8-HCDF) were those that exhibited high cytosolic Ah receptor binding affinities (1.5 – 7.5 × 10 −8 M) and were also potent AHH inducers. The inactive compound (1,2,4,7,8-PeCDF) was less active in the receptor binding and enzyme induction assays. The results showed that there was a structure-dependent correlation between the Ah receptor agonist activity of a congener and its potency to reduce I-compounds in female Sprague-Dawley rat liver.

Ageing has no effect on the volume density of hepatocytes, reticulo-endothelial cells or the extracellular space in livers of female Sprague-Dawley rats.

1. The hepatic reticulo-endothelial cell population is generally assumed to increase in size, along with the liver, during ageing in rats. However, this has not been rigorously established. 2. Using electron microscopy and stereological techniques, the present study has shown that the volume densities of hepatocytes and Kupffer cells (and probably also of endothelial cells, fat storing cells and the extracellular space) of the livers of female Sprague-Dawley rats are the same at 2 and 24-25 months of age. 3. This result indicates that the increase in size of the liver during ageing in the rat is associated with an equivalent increase in the volume of each cell population and the extracellular space.

Hepatotoxicity and carcinogenicity in female Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD): A pathology working group reevaluation

Risk assessment for 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) has been based in part on the incidences of liver neoplasms in female Sprague-Dawley (SD) rats reported in a 2-year study conducted by Dow Chemical Corporation and published in 1978. In the years subsequent to the Dow report, the criteria for the diagnosis of proliferative hepatocellular lesions in the rat have been refined based upon ongoing study of these lesions. Because of this, PATHCO, Inc., was requested to conduct an independent review of the liver slides from the Dow TCDD study in order to assess how the current terminology might impact on interpretation of proliferative liver lesions in rats compared to the terminology used in the past. In March 1990, a pathology working group (PWG) was convened to review proliferative lesions in the livers of the female rats. The results of the PWG's evaluation of the microslides indicated a trend in tumor incidence similar to that published in 1978 but with a lower incidence of tumors in the middle and high dose females. Based on the morphologic findings, including the fact that the tumors were predominantly benign and usually associated with lesions of hepatic toxicity, the PWG considered this study to demonstrate a weak oncogenic effect of TCDD in the livers of female SD rats. As a result of its review, the PWG noted that in order to establish a relationship between the toxic hepatitis and the hepatocellular neoplasms, an independent review and grading of the toxic lesions in all female rats would be required. This subsequent review found that in the middle and high dose groups, tumors were observed only in animals with toxicity. In the low dose animals, there was an increase in the incidence and multiplicity of eosinophilic foci and a slight increase in the incidence of hepatotoxicity of a minimal degree of severity compared to controls, but there was no increase in the incidence of hepatocellular neoplasms. There appeared to be a distinct correlation between the presence of overt hepatotoxicity and the development of hepatocellular neoplasms.