Interactive effects of dietary adaptation period length and titration diet type on apparent ileal phosphorus digestibility and phosphorus retention in growing broilers.

Two experiments were conducted to examine the effects of different corn titration diets and dietary adaptation period length (DAPL) on intestinal histology, apparent ileal P digestibility (AIPD), and apparent P retention (APR) in Ross × Ross 708 male broilers from 20 to 24 d of age. It was hypothesized that purified ingredients in nutrient-deficient titration diets may affect P availability with varying DAPL. In experiment 1, 1,152 broilers were utilized in a 3 × 3 factorial treatment structure with 3 diets (control, 25% corn titration diet [25CTD], or 75% corn titration diet [75CTD]) and 3 DAPL (0, 24, or 72 h). Experiment 2 was conducted with 576 broilers as a 4 × 3 factorial arrangement with 4 diets (control, 25CTD, 75CTD, or nitrogen-free diet [NFD]) and 3 DAPL (24, 48, or 72 h). All diets contained purified ingredients except for the control diet, which had the same formulation as the common starter and served as a control for DAPL. The NFD diet was fed as a highly purified protein-free diet. Broilers were fed a common diet until 19 d of age and then transferred to experimental diets at 20 d of age. In experiment 1, diet type did not affect (P > 0.05) intestinal histology. However, diet type and DAPL each influenced (P.≤.0.001) diet AIPD. Higher (P.≤.0.001) AIPD was measured for the control diet compared with the 75CDT, and the 25CTD had the lowest AIPD. Following a 24 h DAPL, AIPD was higher (P.≤.0.001) than after a DAPL of 0 or 72 h. In experiment 2, diet type × DAPL interactions (P.≤.0.001) were observed for APR of the control diet, 75CTD, and NFD, but not the 25CTD. Because APR of the control diet was affected by varying DAPL, factors other than differences in diet type may have been responsible for inconsistencies in the measure of P availability. Furthermore, no clear evidence was observed that broilers were able to adapt to P-deficient diets by increasing APR or AIPD. In conclusion, a standard DAPL should be established as a means to reduce variability associated with measuring of feedstuff P availability.

Experimental transmission of Corynebacterium pseudotuberculosis biovar equi in horses by house flies.

BACKGROUND: The route of Corynebacterium pseudotuberculosis infection in horses remains undetermined, but transmission by insects is suspected. OBJECTIVES: To investigate house flies (Musca domestica L.) as vectors of C. pseudotuberculosis transmission in horses. ANIMALS: Eight healthy, adult ponies. METHODS: Randomized, controlled, blinded prospective study. Ten wounds were created in the pectoral region where cages for flies were attached. Three ponies were directly inoculated with C. pseudotuberculosis. Four ponies were exposed for 24 hours to 20 hours C. pseudotuberculosis-inoculated flies. One negative control pony was exposed to noninoculated flies. Ponies were examined daily for swelling, heat, pain, and drainage at the inoculation site. Blood was collected weekly for CBC and biochemical analysis, and twice weekly for synergistic hemolysis inhibition titers. RESULTS: Clinical signs of local infection and positive cultures were observed in 7/7 exposed ponies and were absent in the negative control. In exposed ponies, peak serologic titers (1:512 to 1:2,048) were obtained between days 17 and 21. Seroconversion was not observed in the negative control. Neutrophil counts were higher in the positive and fly-exposed groups than in the negative control (P = .002 and P = .005) on day 3 postinoculation. Serum amyloid A concentrations were higher in the positive control than in the negative control and fly-exposed ponies on days 3 (P < .0001) and 7 (P = .0004 and P = .0001). No differences were detected for other biochemical variables. CONCLUSIONS AND CLINICAL IMPORTANCE: House flies can serve as mechanical vectors of C. pseudotuberculosis and can transmit the bacterium to ponies.

Hepatocellular proliferation in response to a peroxisome proliferator does not require TNFalpha signaling.

Rodents exposed to peroxisome proliferator xenobiotics respond with marked increases in hepatocellular replication and growth that results in tumor formation. Recently, tumor necrosis factor-alpha (TNFalpha) was proposed as the central mediator of this maladaptive response. To define the role of TNFalpha signaling in hepatocellular growth induced by peroxisome proliferators we administered three daily gavage doses of the potent peroxisome proliferator, Wy-14 643, to mice nullizygous for TNF-receptor I (TNFR1), TNFR2, or both receptors. We demonstrate here that regardless of genotype the mice responded with almost identical increases in liver to body weight ratios and hepatocyte proliferation. Lacking evidence that TNFalpha signaling mediates these effects, we then examined the possible contribution of alternative cytokine pathways. Semi-quantitative, reverse transcriptase polymerase chain reaction analysis revealed that wild type mice acutely exposed to Wy-14 643 had increased hepatic expression of Il1beta, Il1r1, Hnf4, and Stat3 genes. Moreover, hepatic adenomas from mice chronically exposed to Wy-14 643 had increased expression of Il1beta, Il1r1, Il6, and Ppargamma1. Expression of Il1alpha, Tnfalpha, Tnfr1, Tnfr2, Pparalpha, or C/ebpalpha was not altered by acute Wy-14 643 exposure or in adenomas induced by Wy-14643. These data suggest that the hepatic mitogenesis and carcinogenesis associated with peroxisome proliferator exposure is not mediated via TNFalpha but instead may involve an alternative pathway requiring IL1beta and IL6.

IL-18-binding protein protects against lipopolysaccharide- induced lethality and prevents the development of Fas/Fas ligand-mediated models of liver disease in mice.

IL-18-binding protein (IL-18BP) is a natural IL-18 inhibitor. Human IL-18BP isoform a was produced as fusion construct with human IgG1 Fc and assessed for binding and neutralizing IL-18. IL-18BP-Fc binds human, mouse, and rat IL-18 with high affinity (K(D) 0.3-5 nM) in a BIAcore-based assay. In vitro, IL-18BP-Fc blocks IL-18 (100 ng/ml)-induced IFN-gamma production by KG1 cells (EC(50) = 0.3 microg/ml). In mice challenged with an LD(90) of LPS (15 mg/kg), IL-18BP-Fc (5 mg/kg) administered 10 min before LPS blocks IFN-gamma production and protects against lethality. IL-18BP-Fc administered 10 min before LPS blocks IFN-gamma production induced by LPS (5 mg/kg) with ED(50) of 0.005 mg/kg. Furthermore, IL-18BP-Fc (5 mg/kg) abrogates LPS (5 mg/kg)-induced IFN-gamma production even when administered 6 days before LPS but shows no effect when administered 9 or 12 days before LPS. Given 10 min before LPS challenge to mice primed 12 days in advance with heat-killed Propionibacterium acnes, IL-18BP-Fc prevents LPS-induced liver damage and IFN-gamma and Fas ligand expression. Given at the moment of priming with P. acnes, IL-18BP-Fc decreases P. acnes-induced granuloma formation, macrophage-inflammatory protein-1alpha and macrophage-inflammatory protein-2 production and prevents sensitization to LPS. IL-18BP-Fc also prevents Con A-induced liver damage and IFN-gamma and Fas ligand expression as well as liver damage induced by Pseudomonas aeruginosa exotoxin A or by anti-Fas agonistic Ab. In conclusion, IL-18BP can be engineered and produced in recombinant form to generate an IL-18 inhibitor, IL-18BP-Fc, endowed with remarkable in vitro and in vivo properties of binding and neutralizing IL-18.

Oxidants from nicotinamide adenine dinucleotide phosphate oxidase are involved in triggering cell proliferation in the liver due to peroxisome proliferators.

It was shown that 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643), a potent peroxisome proliferator, caused rapid oxidant-dependent activation of nuclear factor kappaB (NF-kappaB) in Kupffer cells in vivo and activated superoxide production by isolated Kupffer cells. Here, we tested the hypothesis that NADPH oxidase (NADPH OX) is the source of oxidants increased by Wy-14,643. Indeed, both activation of NF-kappaB and increases in cell proliferation due to a single dose of Wy-14,643 (100 mg/kg) were prevented completely when rats were pretreated with diphenyleneiodonium (1 mg/kg), an inhibitor of NADPH OX. p47phox is a critical subunit of NADPH OX; therefore, p47phox knockout mice were used to specifically address the hypothesis of NADPH OX involvement. In livers of wild-type mice, Wy-14,643 activated NF-kappaB, followed by an increase in mRNA for tumor necrosis factor a. Importantly, these changes did not occur in p47phox knockouts. Moreover, when Kupffer cells were treated with Wy-14,643 in vitro, superoxide production was increased in cells from wild-type but not p47phox-null mice. Finally, when mice were fed a Wy-14,643-containing (0.1%) diet for 7 days, the increase in liver weight and cell proliferation caused by Wy-14,643 in wild-type mice was blocked in p47phox-null mice. Combined, these results are consistent with the hypothesis that Wy-14,643 activates NADPH OX, which leads to NF-kappaB-mediated production of mitogens that causes hepatocellular proliferation characteristic of this class of nongenotoxic carcinogens.

Pubertal development and reproductive functions of Crl:CD BR Sprague-Dawley rats exposed to bisphenol A during prenatal and postnatal development.

Bisphenol A (BPA) is used on a large scale in the manufacture of polycarbonate plastics. BPA has been shown to bind weakly to both estrogen receptor (ER)alpha and ERbeta, and to transactivate reporter genes in vitro. The purpose of the present study was to determine whether exposure of rats to BPA during pre- and postnatal development affects estrogen-mediated end points related to pubertal development and reproductive functions. BPA was administered to pregnant Crl:CD BR Sprague-Dawley rats by gavage at 0, 3.2, 32, or 320 mg/kg/day from gestation day (GD) 11 through postnatal day (PND) 20. Diethylstilbestrol (DES) at 15 microg/kg/day was used as a reference chemical with known estrogenic effects. Female pubertal development was not affected by indirect BPA exposure of the offspring at any of the dose levels. Treatment with this chemical also did not produce detectable effects on the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA), estrous cyclicity, sexual behavior, or male reproductive organ weights of F(1) offspring. However, DES at 15 microg/kg/day increased the volume of the SDN-POA of female offspring and affected their normal estrous cyclicity following puberty. In this study, pre- and postnatal exposure of rats to BPA at 3.2, 32, or 320 mg/kg/day from GD 11 through PND 20 did not have any apparent adverse effects on female rat pubertal development and reproductive functions.

Dose-dependent alterations in androgen-regulated male reproductive development in rats exposed to Di(n-butyl) phthalate during late gestation.

Di(n-butyl) phthalate (DBP) is a commercially important plasticizer and ubiquitous environmental contaminant. Since previous, limited dose-response studies with DBP that reported alterations in male reproductive development and function failed to establish a NOAEL (no-observed-adverse-effect level), an extensive dose-response study was conducted. Pregnant CD rats were given DBP by gavage at 0, 0.5, 5, 50, or 100 mg/kg/day (n = 19-20) or 500 mg/kg/day (n = 11) from gestation day 12 to 21. In male offspring, anogenital distance was decreased at 500 mg DBP/kg/day. Retained areolas or nipples were present in 31 and 90% of male pups at 100 and 500 mg/kg/day, respectively. Preputial separation was not delayed by DBP treatment in males with normal external genitalia, but cleft penis (hypospadias) was observed in 5/58 rats (4/11 litters) at 500 mg/kg/day. Absent or partially developed epididymis (23/58 rats in 9/11 litters), vas deferens (16/58 animals in 9/11 litters), seminal vesicles (4/58 rats in 4/11 litters), and ventral prostate (1/58 animals) occurred at 500 mg/kg/day. In 110-day-old F(1) males, the weights of the testis, epididymis, dorsolateral and ventral prostates, seminal vesicles, and levator ani-bulbocavernosus muscle were decreased at 500 mg/kg/day. At 500 mg/kg/day, widespread seminiferous tubule degeneration was seen in 25/58 rats (in 9/11 litters), focal interstitial cell hyperplasia in 14/58 rats (in 5/11 litters), and interstitial cell adenoma in 1/58 rats (in 1/11 litters). For this 10-day prenatal (embryonic and fetal) exposure to DBP, the NOAEL and LOAEL (lowest-observed-adverse-effect level) were 50 and 100 mg/kg/day, respectively. This is currently the lowest NOAEL described for the toxicity of DBP.

Apoptosis, mitosis and cyclophilin-40 expression in regressing peroxisome proliferator-induced adenomas.

Chronic exposure to peroxisome proliferators (PP), including certain industrial and pharmaceutical chemicals, causes liver cancer in rodents. Continuous exposure to PP is needed for tumor development since the frequency of hepatocellular neoplasms is decreased in animals returned to control diet. To determine cellular and molecular events responsible for enhanced growth in PP-induced liver tumors, we evaluated the relationships of WY-14,643 levels, apoptosis, mitosis and cyclophilin-40 (Cyp-40) expression in regressing tumors induced by WY-14,643, a potent PP. Male F344 rats were fed WY-14,643 (0.1%) in the diet for 43 weeks and then switched to control diet for 2, 3, 5 or 36 days. Mean serum and hepatic concentrations of WY-14,643 were decreased as early as 2 days following removal of WY-14,643 as compared with rats continuously fed WY-14,643. Adenomas from rats maintained on WY-14,643 markedly compressed surrounding parenchyma. Evidence of adenoma regression was observed by 3 days of WY-14,643 withdrawal and was characterized by loss of compression. Decreased compression corresponded to increases in the apoptotic index and decreases in the mitotic index in regressing adenomas at 2, 3, and 5 days following the switch to control diet. Cyclophilins are multifunctional receptor proteins involved in numerous signal transduction pathways, including those mediated by cyclosporin, a liver tumor promoter in rats. Cyp-40 expression was markedly increased in adenomas from continuously exposed rats, but expression returned to levels similar to surrounding parenchyma in adenomas after 5 days of WY-14,643 withdrawal. Taken together, these results indicate that WY-14, 643-induced adenomas regress rapidly following withdrawal of the PP in association with declining liver WY-14,643 levels, suggesting that peroxisome proliferator-activated receptor alpha may mediate PP-induced alterations in mitogenic and/or apoptotic regulation in growing tumors, in conjunction with alterations in Cyp-40 signal transduction.

Alterations in endocrine responses in male Sprague-Dawley rats following oral administration of methyl tert-butyl ether.

Methyl tert-butyl ether (MTBE) is an oxygenated fuel additive used to decrease carbon monoxide emissions during combustion. MTBE is a nongenotoxic chemical that induces Leydig cell tumors (LCT) in male rats. The mechanism of MTBE-induced LCT is not known; however, LCT induced by other nongenotoxic chemicals have been associated with the disruption of the hypothalamus-pituitary-testicular (HPT) axis. The objective of this study was to determine whether MTBE functions as an endocrine-active compound by affecting levels of specific hormones involved in the maintenance of the HPT axis. Nine-week-old male Sprague-Dawley rats were administered MTBE by gavage at 0, 250, 500, 1000, or 1500 mg MTBE/kg/day for 15 or 28 consecutive days and sacrificed 1 h following the last dose. Relative testis weights were increased only in high-dose animals treated for 28 days, and no testicular lesions were observed at any dose level. Adrenal gland, liver, and kidney weights were also increased. Histologic changes included protein droplet nephropathy of the kidney and centrilobular hypertrophy of the liver. Interstitial fluid and serum testosterone levels as well as serum prolactin levels were decreased only in animals treated with 1500 mg MTBE/kg/day for 15 days. At 28 days, serum triiodothyronine (T3) was significantly decreased at 1000 and 1500 mg MTBE/kg/day compared to control animals, and a decrease in serum luteinizing hormone and dihydrotestosterone was observed at 1500 mg MTBE/kg/day. These results indicate that MTBE causes mild perturbations in T3 and prolactin; however, the changes in testosterone and LH levels did not fit the pattern caused by known Leydig cell tumorigens.

Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment.

The National Toxicology Program (NTP) conducted a continuous breeding study in SD rats with di-n-butyl phthalate (DBP) given via the diet at dose levels of up to 650 mg/kg/day. In the parental generation effects on reproduction were modest (small decreases in litter size and pup weight following treatment). However, the F(1) male offspring had marked decreases in fertility (at 650 mg/kg/day), with reduced sperm counts and reproductive tract malformations on reaching adulthood. A no-observed-adverse-effect level (NOAEL) was not established for the study [lowest-observed-adverse-effect level (LOAEL) 66 mg/kg/day]. In a study conducted at CIIT, the majority of these adverse changes could be reproduced over a similar dose range, but with a much shorter dosing regimen covering a critical window of development (gestation days 12-20). A default risk assessment for DBP indicates a reference dose (RfD) of 66 microg/kg/day, based on a LOAEL of 66 mg/kg/day and default factors of 10 for inter-species and inter-individual differences and the lack of a NOAEL. Human exposure data would indicate worst-case scenarios to infants (via formula) in the dose range of the RfD. A default risk assessment appears to be inappropriate since rodents, unlike primates, metabolize phthalate diesters (including DBP) to monoesters extensively in the gut following oral administration. It is believed that the monoester is the active principle for induction of reproductive and developmental toxicity of specific phthalate esters. Thus, if humans produce very low levels of the monoester from an environmental exposure to the diester, the likelihood of any reproductive or developmental toxicity via the oral route appears extremely remote.

Dietary glycine prevents the development of liver tumors caused by the peroxisome proliferator WY-14,643.

Previous studies demonstrated that dietary glycine prevents elevated rates of cell proliferation following treatment with the peroxisome proliferator and liver carcinogen WY-14,643. Since increased cell replication is associated with the development of hepatic cancer caused by peroxisome proliferators, glycine may have anti-cancer properties. Therefore, experiments were designed to test the hypothesis that dietary glycine would inhibit the hepatocarcinogenic effect of WY-14,643. Male F344 rats were fed four different NIH 07-based diets: 5% glycine; 5% valine for nitrogen balance (control); 0.1% WY-14,643 + 5% valine (WY-14,643); 0.1% WY-14,643 + 5% glycine (WY-14,643 + glycine). Food consumption did not differ among the groups, but WY-14,643-fed rats weighed 10-25% less than expected based on previous studies. Serum glycine levels were elevated 4-5-fold by glycine-containing diets; however, the 10-fold increase in peroxisomal enzyme activity caused by WY-14,643 was unaffected by the addition of 5% glycine to the diet. After 22 weeks, livers from rats fed WY-14,643 had a similar incidence and multiplicity of proliferative lesions (foci and adenomas) to those fed WY-14,643 + glycine. Moreover, cell proliferation in the surrounding 'normal' parenchyma (labeling index approximately 4%) and foci (labeling index approximately 50%) did not differ between WY-14,643 and WY-14,643 + glycine-fed rats. However, after 51 weeks of dietary exposure to WY-14,643, glycine prevented formation of small (0-5 mm diameter) tumors by 23% and inhibited the development of medium size (5-10 mm) tumors by 64%. Furthermore, glycine prevented the formation of the largest tumors (>10 mm) by nearly 80%. Thus, glycine did not inhibit early foci formation; however, it significantly decreased their ability to progress to tumors. Moreover, the inhibitory effect of glycine was greater with increasing tumor size. These studies demonstrate that dietary glycine prevents the development of hepatic tumors caused by the peroxisome proliferator WY-14,643 consistent with the idea that it may be an effective chemopreventive agent.

Hepatic expression of acute-phase protein genes during carcinogenesis induced by peroxisome proliferators.

Concern exists regarding peroxisome proliferator (PP) xenobiotic exposure because many PPs are potent hepatocarcinogens in rodents. The mechanism of carcinogenicity induced by PPs is atypical compared with those of other hepatocarcinogens in that the former appears to involve alterations in expression of PP-activated receptor (PPAR) target genes rather than direct mutagenicity. To begin to identify some of these genes, we used differential display to compare mRNA expression between hepatic adenomas and adjacent non-tumor liver from rats fed the potent PP Wy-14643 (WY) for 78 wk. Here, we report increased expression of the acute-phase protein (APP) gene alpha-1 antitrypsin (AT) and decreased expression of alpha2-urinary globulin in the tumors. Similar changes were seen in hepatic adenomas induced by a diethylnitrosamine and phenobarbital protocol, indicating a lack of specificity for PP-induced tumors. Additional APP genes, including ceruloplasmin, haptoglobin, beta-fibrinogen, and alpha1-acid glycoprotein were also upregulated in WY-induced tumors but were downregulated in the livers of rats administered a different PP for 13 wk. Mice treated with either WY or di(2-ethylhexyl) phthalate for 3 wk had decreased hepatic AT expression but increased expression of ceruloplasmin and haptoglobin. PPARalpha-null mice showed no hepatic APP gene alteration after PP treatment but had higher basal expression than did wild-type controls. We conclude that PPARalpha activation by several different PPs leads to dysregulation of hepatic APP gene expression in rats and mice. This dysregulation may indicate alterations in cytokine signaling networks regulating both APP gene expression and hepatocellular proliferation.

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Dysregulation of apoptosis by c-myc in transgenic hepatocytes and effects of growth factors and nongenotoxic carcinogens.

Regulation of apoptosis is an important component of multistage hepatocarcinogenesis. The proto-oncogene c-myc has been shown to be important in apoptosis regulation and to be amplified and overexpressed in human and rodent liver neoplasia. The objectives of the study reported here were to determine whether apoptosis regulation is altered in transgenic hepatocytes that overexpress c-myc and whether growth factors or nongenotoxic carcinogens alter apoptosis regulation in c-myc versus wild-type hepatocytes. Hepatocytes isolated from c-myc transgenic mice had four fold more c-myc RNA and protein (at 12-48 h) in addition to increased apoptosis levels compared with wild-type hepatocytes. The increased apoptosis in c-myc hepatocytes was accompanied by increased p53, bax, and bak and decreased bcl-2 protein levels. Hepatocytes overexpressing c-myc were more sensitive to apoptosis induced by bleomycin but less sensitive to apoptosis induced by transforming growth factor (TGF)-beta. Phenobarbital, a potent liver tumor promoter, inhibited apoptosis in c-myc hepatocytes but not in wild-type hepatocytes, decreased p53 and bax, and increased bcl-2 protein levels. Nafenopin inhibited apoptosis in both c-myc and wild-type hepatocytes, whereas 2,3,7,8-tetrachlorodibenzo-pdioxin did not inhibit apoptosis in either wild-type or c-myc hepatocytes. TGF-alpha inhibited apoptosis and increased bcl-X(L) and decreased bak protein levels in c-myc hepatocytes but not in wild-type hepatocytes. Insulin-like growth factor-II did not affect apoptosis in c-myc or wild-type hepatocytes. In this study, overexpression of c-myc altered the response to apoptotic stimuli in transgenic hepatocytes. Furthermore, phenobarbital and TGF-alpha inhibited c-myc-induced apoptosis, which may have resulted in a selective growth advantage for an initiated cell population and which may be a mechanism for tumor promotion.

Altered bcl-2 family expression during non-genotoxic hepatocarcinogenesis in mice.

Dysregulation of apoptosis is an important component of multistage hepatocarcinogenesis. Members of the bcl-2 protein family are important in the regulation of apoptosis and their expression is altered in several cancers. The objectives of the present study were to determine whether the expression of members of the bcl-2 protein family are altered in mouse liver during acute treatment with non-genotoxic carcinogens and throughout non-genotoxic hepatocarcinogenesis. Acute treatment of B6C3F1 mice with phenobarbital resulted in increased levels of bcl-2 and decreased levels of bax protein, while acute treatment with WY-14,643 resulted in increased bcl-2 and BAG-1 protein in the liver. Following chronic treatment, altered hepatic foci and adenomas were classified as: small-cell, heterogeneous basophilic lesions (spontaneous or tetrachlorodibenzo-p-dioxin-induced); large-cell, homogeneous basophilic lesions (WY-14,643-induced); acidophilic lesions (phenobarbital- or chlordane-induced). Of the small-cell heterogeneous basophilic lesions, 86% of foci (31/36) and 85% of adenomas (35/41) exhibited increased bcl-2 protein levels compared with surrounding normal hepatocytes, whereas only 12.5% of foci (4/36) and 12% of adenomas (5/41) exhibited increased bcl-X(L) levels. Of the large-cell, homogenous, basophilic lesions, 100% of foci (3/3) and 90% of adenomas (9/10) expressed bcl-2 protein, whereas 100% of foci (3/3) and 80% of adenomas (8/10) exhibited increased bcl-X(L) protein levels compared with surrounding normal hepatocytes. Of the acidophilic lesions, the majority of foci (28/32, 88%) and adenomas (47/50, 94%) expressed increased bcl-X(L), whereas increased bcl-2 was observed in only 12.5% of acidophilic preneoplastic foci (4/32) and 14% of acidophilic adenomas (7/50). Of the carcinomas analyzed, 81% expressed increased bcl-2 (54/67), 78% expressed increased bcl-X(L) (52/67) and 69% expressed increased levels of both bcl-2 and bcl-X(L) (46/67). Collectively, only 8% of preneoplastic foci, 3% of adenomas and 1.5% of carcinomas did not express either bcl-2 or bcl-X(L). These results suggest that regulation of apoptotic proteins is altered during non-genotoxic carcinogenesis in mouse liver. Furthermore, there were both chemical- and lesion-specific aspects of expression of apoptotic proteins during hepatocarcinogenesis in mice.

Manganese-induced developmental neurotoxicity in the CD rat: is oxidative damage a mechanism of action?

Inhalation of high concentrations of manganese (Mn) is associated with an extrapyramidal motor disorder in humans. Oxidative damage, mediated by increased levels of Mn in dopaminergic brain regions and mitochondria, is a hypothesized mechanism of action for Mn-induced neuronal degeneration and loss. To test this proposed mechanism, developing CD rats, which may be at an increased risk for Mn-induced neurotoxicity, were exposed orally to 0, 25, or 50 mg/kg/day of MnCl2 from postnatal day (PND) 1 to 49 Brain regional and mitochondrial Mn levels, brain regional reactive oxygen species (ROS) levels, and whole-brain nuclear and mitochondrial 8-OHdG levels were used to evaluate Mn-mediated oxidative damage. High-dose Mn exposure was associated with increased spontaneous motor activity on PND 21 and decreased body weights on PND 49. On PND 21, Mn concentrations were increased in brain regions and mitochondrial fractions in both low- and high-dose groups. ROS levels were elevated in cerebellum but not striatum. On PND 49, Mn concentrations in brain regions and mitochondrial fractions were increased only in the high-dose group. Mn exposure did not significantly alter 8-OHdG levels in either mitochondrial or nuclear DNA. Selective uptake of Mn by the striatum or mitochondrial fraction was not demonstrated at either time point. These data allow us to conclude that oral exposure to high levels of Mn in developing CD rats resulted in increased brain regional and mitochondrial Mn levels, increased motor activity, and decreased body weights but not in selective accumulation of Mn in the striatum or mitochondrial fraction of any brain region or elevations in striatal ROS or whole-brain 8-OHdG levels. These findings do not support the hypothesis that oxidative damage, as assessed by ROS and 8-OHdG levels, is a mechanism of action in Mn-induced developmental neurotoxicity in the CD rat.

Alteration of protein kinase C isoform-specific expression during rat hepatocarcinogenesis after exposure to the peroxisome proliferator WY-14,643.

The role of protein kinase C (PKC) isoforms in mediating peroxisome proliferator chemical- (PPC) induced hepatocarcinogenesis was examined. After an acute gavage exposure to WY-14,643 (WY) membrane-bound PKCdelta and cytosolic PKCbeta decreased, whereas the expression of the other isoforms was not altered. After a 13-week chronic exposure, membrane-bound PKCbeta, delta and zeta levels decreased. In WY-induced hepatocellular adenomas, PKCalpha was increased, and PKCbeta was further decreased in membrane fractions. These results, taken together with previous studies, indicate that alterations in PKCalpha, beta and delta isoforms, which regulate mitogenesis, could play important roles in perpetuating the high cell proliferative rate in PPC-induced hepatocellular adenomas.