Activity of lysosomal system in mouse liver after taxol administration

• 1.1. The effect of taxol on selected lysosomal enzymes (cathepsin D, lysosomal lipase, β-glucuronidase, β-glucosidase, alanine aminopeptidase) in mouse hepatocytes after 24-hr treatment by increasing doses (0.75 mg/kg bw, 1.25 mg/kg bw and 2.5 mg/kg bw) was studied.
• 2.2. The segments were also taken from the mice for ultrastructural studies with the use of electron microscopy. The greatest changes in activity of enzymes at the taxol dose of 2.5 mg/kg bw were as follows: the activity of cathepsin D increased by 71%, that of alanine aminopeptidase increased by 103%, that of β-glucuronidase decreased by 45% and that of β-glucosidase decreased by 63%.
• 3.3. The significant changes observed in the hepatocyte ultrastructure were closely correlated with biochemical changes that were dependent on the taxol dosage.

     

Oxidative stress in female B6C3F1 mice following acute and subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23-27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15-150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001-100 microg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 microg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following sub-chronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.     

Effects of cypermethrin, propetamphos, and combination involving cypermethrin and propetamphos on lipid peroxidation in mice

Insecticides are the chemicals widely used in agriculture, environmental health, human-and animal-health fields. Exposure to insecticides has been associated with many hazardous effects, including antioxidative metabolism. In the current study, the effect of cypermethrin (CYP), propetamphos (PRO) and their mixtures on oxidative stress in mice to understand the possible health effects to animals and human beings was investigated. In the present study, 245 male Albino mice weighing 35-40 g were used. The mice were divided into seven groups. The first group served as the control group. The second and third groups were administered CYP at doses of 5 mg/kg/bw and 10 mg/kg/bw, respectively, and the fourth and fifth groups were given PRO at doses of 2.5 mg/kg/bw and 5.0 mg/kg/bw, respectively. The sixth and seventh groups received combination regimens containing 5 mg/kg/bw CYP plus 2.5 mg/kg/bw PRO and 10 mg/kg/bw CYP plus 5 mg/kg/bw PRO, respectively, in feed for 60 days. Blood samples were collected by cardiac puncture on the 15th, 45th and 60th days. Serum nitric oxide (NO) and plasma malondialdehyde (MDA) levels and erythrocyte superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were measured. In conclusion, the alterations observed in the MDA and NO levels and SOD, CAT, and GSH-Px activities of the trial groups, demonstrate the administration of certain doses of CYP and PRO, either alone or combined, to mice for a period of 60 days to produce oxidative stress. The degree of oxidative stress was found to be related to the dose administered, the duration of exposure and the administration of the indicated compounds either alone or as a combination.     

A comparison of a commercial polybrominated biphenyl mixture, 2,4,5,2',4',5'-hexabromobiphenyl and 2,3,6,7-tetrabromonaphthalene as inducers of liver microsomal drug-metabolizing enzymes.

The commercial polybrominated biphenyl (PBB) mixture, Firemaster BP-6, is a mixed inducer of hepatic drug-metabolizing enzymes in the rat. Its effects resemble those of a combination of the phenobarbital and 3-methylcholanthrene classes of inducers. 2,3,6,7-Tetrabromonaphthalene (TBN) was studied as a prototype of the brominated naphthalenes which are present as minor contaminants of Firemaster BP-6. TBN is an isostere of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent known inducer of the 3-methylcholanthrene class. TBN is a 3-methylcholanthrene-type inducer; however, it is 10⁴ times less potent than TCDD and it is only slightly more potent than Firemaster. The relatively low potency of TBN does not appear to be due to metabolism, since comparable amounts of TBN and Piremaster were found in the liver at equivalent doses. In contrast to TBN, the predominant component, 2,4,5,2',4',5'-hexabromobiphenyl (HBB), is a pure phenobarbital-type inducer. The no effect level for the effects of a single dose of Firemaster or 2,4,5,2',4',5'-HBB on hepatic microsomal enzymes was 8 μmoles/kg (4.7 mg/kg of Firemaster). When Firemaster was given chronically 5 days a week for 15–30 days, changes in hepatic enzymes occurred with doses as low as 0.3 mg/kg/day. Using liver enzyme activities as an index of hepatic change, a 30-day recovery study showed that the liver does recover partially after exposure ceases. The degree of recovery correlates with a decrease in the concentration of PBBs in the liver as PBBs are redistributed from the liver to the fat. Porphyria was not observed during the chronic experiment, but gross hepatic porphyria developed in the Firemaster-treated rats during the recovery period.     

Dietary toxicity of picloram herbicide in rats

The toxicity of orally administered technical-grade picloram was evaluated in male and female Fischer 344 rats. Dietary dose levels were up to 2000 mg/kg body weight (bw) X d for 2 wk, 500 mg/kg bw X d for 13 wk, or 200 mg/kg bw X d for 12 mo. Routine indices of toxicity were evaluated at all of the respective time periods. Body weight, food consumption, clinical chemistries, urinalyses, and hematological determinations were considered unaffected by treatment. The only treatment-related effect, regardless of the duration of exposure, was in the liver of both male and female rats. This was generally manifested as an increase in the liver-to-body weight ratio and slight hypertrophy and pallor of the centrilobular hepatocytes. These effects were consistently present in rats receiving 1000 mg/kg bw X d for 2 wk, 300 mg/kg bw X d for 13 wk, or 200 mg/kg bw X d for 6 or 12 mo. Similar effects were marginally evident for rats receiving 500 mg/kg bw X d for 2 wk, 150 mg/kg bw X d for 13 wk, or 60 mg/kg bw X d for 6 or 12 mo. At 60 mg/kg bw X d, the effects were not progressive from 6 to 12 mo. The no-observable-effect level (NOEL) was 20 mg/kg bw X d for male and female rats fed picloram for 12 mo.     

Influence of the Ah locus on the humoral immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: evidence for Ah-receptor-dependent and Ah-receptor-independent mechanisms of immunosuppression

There are conflicting reports in the literature regarding the role of the Ah locus in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) immunotoxicity. The present studies have utilized two congenic strains of C57Bl/6 mice that differ only at this locus to assess its influence on TCDD-induced suppression of antibody responses. Mice were given a single oral dose of TCDD 2 days prior to challenge with sheep red blood cells (SRBC) or trinitrophenyl-lipopolysaccharide (TNP-LPS). The subsequent dose-dependent effects of TCDD on the amount of antibody produced by splenic plasma cells were measured using the hemolytic antibody isotope release assay. In addition, the relative importance of the Ah genotype of lymphoid versus nonlymphoid tissue was examined in adoptive transfer experiments. Aryl hydrocarbon hydroxylase (AHH) activity was significantly induced in Ahbb mice by a dose of 0.5 micrograms/kg TCDD and maximally induced by a dose of 2 micrograms/kg. Ahdd mice required 10-fold higher doses of TCDD to induce comparable levels of AHH. The degree of thymic involution and liver hypertrophy induced by TCDD was also influenced by the Ah genotype of the animals. Both Ahbb and Ahdd mice exhibited dose-dependent suppression of the anti-TNP response following TCDD exposure. The ID50 was 7.0 micrograms/kg in Ahbb mice and 30.8 micrograms/kg in Ahdd mice. Suppression of the antibody response to SRBC was also dependent on the Ah locus. The ID50 in Ahbb mice was 0.6 micrograms/kg TCDD. However, an apparent biphasic dose response for suppression of the anti-SRBC response in Ahdd mice suggested the involvement of an Ah-independent component of suppression as well. In adoptive transfer studies, lymphocytes were identified as an Ah-dependent component of the response. The Ah-independent component of the response was not identified, and could be either lymphoid or nonlymphoid in nature. The possibility that T helper cells represent the Ah-independent component is discussed.     

Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants (HBCD,BDE-47, PCB-153, TCDD): Comparative assessment of effects in potential target tissues

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2′,4,4′-Tetrabromodiphenyl ether BDE-47; 2,2′,4,4′,5,5′-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 μg/kg bw/day; PCB-153 195 μg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.     

Toxicity of TCDD in European flounder (Platichthys flesus) with emphasis on histopathology and cytochrome P450 1A induction in several organ systems

The present study is part of a series of experiments, set up to elucidate the impact of aquatic pollution on fish health in the marine and estuarine environment. In the Dutch coastal and estuarine waters, European flounder (Platichthys flesus) showed a relatively high prevalence of (pre)neoplastic liver lesions and lymphocystis virus disease. The hypothesis of a causal relationship between pollution and these diseases was supported by semi-field experiments. Therefore a series of laboratory experiments was performed to further substantiate causality and identify the xenobiotics that may play a major role in the field. Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are important environmental pollutants. They are highly persistent, highly lipophilic, and have shown to induce several toxic effects in mammalian and non-mammalian species at relatively low concentrations. This report describes a study in which European flounder were orally exposed to the most toxic PCDD congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or to harbor sludge extract under controlled laboratory conditions. The effects on several organs (liver, gills, gastro-intestinal tract, thyroid gland, gonads, spleen and mesonephros) were examined microscopically. Induction and localization of cytochrome P4501A (CYP1A) immunoreactivity, and effects on hepatocyte-proliferation were visualized immunohistochemically. Effects on thymus size were examined by morphometric analysis. Oral exposure of flounder to 0.0125 or 0.3125 μg TCDD/kg bw, or to 0.3125 μg TEQ/kg bw of a harbor sludge extract, weekly for 8 weeks, induced a significant increase in CYP1A immunoreactivity in hepatocytes. Single administration of higher doses (20, 100 and 500 μg/kg bw) of TCDD also induced a significant increase CYP1A immunoreactivity in the endothelium in all organs examined, and in the epithelium of the digestive tract, liver, and mesonephros. Remarkably, strong immunoreactivity was noted in a distinct cell population of the hematopoietic tissue in the mesonephros and spleen, which has not been described in fish previously. Moreover, oral exposure to 20 μgTCDD/kg bw resulted in an increased mitotic activity, and an increased hepatosomatic index was found after exposure to 500 μgTCDD/kg bw. In the thymus only a trend in size reduction was noted, again in the highest dose group. Nevertheless, no marked pathology was detected even in fish exposed to a single dose of 500 μg TCDD/kg body weight. The present experiments show that, under the actual experimental conditions, European flounder is relatively insensitive to the toxic effects of TCDD. However, we assume that exposure to TCDD (and related substances) may promote the development of tumors in the field.     

Immunohistochemical localization of thyroid stimulating hormone induced by a low oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats

We have investigated how a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects thyroid hormone regulation, especially in relation to the localization of thyroid stimulating hormone (TSH) in the pituitary and that of thyroxin (T4) of the thyroid in the rat. Female Sprague-Dawley rats were given a single oral administration of TCDD ranging from 1.0 to 4.0 microg/kg body weight (bw), and then tissue specimens were removed on day 7 post-administration. Thyroid hormone concentrations were measured in serum, and the expression of the TCDD-responsive genes, UDP-glucuronosyltransferase-1 (UGT1) and cytochrome P4501A1 (CYP1A1) were examined in the liver. TCDD administration resulted in an increase in both immunostaining intensity and the number of TSH-positive cells in the anterior pituitary. T4 was found to localize only in the follicular lumen of the thyroid in vehicle-treated control rats, while TCDD administration caused a foamy change in the colloid of some follicles, an indication of accelerating the biosynthesis of T4 in the thyroid. By morphometrical analysis, the ratio of parenchymal/lumenal area of the thyroid was found to increase in response to TCDD. TCDD treatment as low as 2.0 microg TCDD/kg bw induced a significant decrease in both serum total T4 (TT4) and free T4 (FT4) concentrations in the rats, with a significant increase in serum TSH levels in the 4.0 microg TCDD/kg bw rats. Serum total triiodothyronine (TT3) level was unchanged in all groups. The UGT1 gene was significantly induced at a TCDD dose as low as 1.0 microg/kg bw in a dose-dependent manner. TCDD concentrations in the serum, liver and adipose tissues were detected in a dose-related fashion. The present immunohistochemical results clearly support the earlier biochemical findings on the perturbation of the thyroid-pituitary axis by TCDD and suggest that UGT1 is an immediate target of a low TCDD exposure that triggers the perturbation.     

Effect of the herbicide 2,4-dichlorophenoxyacetic acid on uropathogenic Escherichia coli virulence factors

The effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-widely used in the world and mainly excreted by the renal route in exposed humans-were studied on the virulence and surface characteristics of an uropathogenic Escherichia coli strain. When the urine was supplemented with 2,4-D in vitro, the compound significantly reduced the bacterial fimbriation assayed by hemagglutination and surface protein quantification. Protein values decreased from 0.24 mg/g dw to 0.05 or 0.12 mg/g dw by 1 or 0.1 mM 2,4-D treatment, respectively. The effects in vivo were studied in groups of mice challenged intra-urethra with E. coli and exposed by the oral route with three different 2,4-D doses (2.6, 25 or 70 mg/kg bw) during 22 days. Depending on the dose used, the herbicide significantly decreased or removed bacterial cells in mice bladder and kidneys; except in the group treated with the highest dose from the 9th day of treatment. The histological studies showed mononuclear cell infiltration at low doses, and toxic damage in the renal parenchyma at prolonged exposure with higher doses, up to tisular necrosis in the 70 mg/kg bw group after 9 days of treatment. Our investigations performed in an experimental model suggest that short time 2,4-D exposure at low doses could act in prevention of UTI stimulating leukocytic migration and decreasing bacterial fimbriation. On the contrary, high doses and long-term exposure enhanced renal damage resulting in infection recurrence.     

Subcellular Localization of TCDD Differs between the Liver, Lungs, and Kidneys after Acute and Subchronic Exposure: Species/Dose Comparisons and Possible Mechanism

Subcellular localization of 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) and related compounds has been examined only in the liver.The objective of this study was (1) to examine and compare thesubcellular distribution of TCDD within hepatic and nonhepatic(lungs/kidneys) tissues of female Sprague-Dawley rats acutelyexposed to TCDD, (2) to analyze species comparisons in the subcellularlocalization of TCDD in multiple tissues, (3) to investigatethe effect of dose on subcellular distribution of TCDD, (4)to analyze the effect of subchronic exposure on the subcellulardistribution of TCDD, and (5) to examine one possible mechanismfor subcellular localization of TCDD. Female Sprague-Dawleyrats and B6C3F1 mice received a single oral dose of 0.1, 1.0,or 10 µg [³H]TCDD/kg body weight and subcellular fractionsof the liver, lungs, and kidneys were prepared by differentialcentrifugation 3 days after exposure. Analysis of the rat subcellularfractions revealed that TCDD was equally distributed betweenthe hepatic P9 (mitochondrial, lysosomal, and nuclear) and S9(cytosol and microsomal) fractions at all doses tested. In contrast,TCDD was concentrated in the P9 of rat nonhepatic tissues atall doses studied. Differential centrifugation of the hepaticS9 showed that TCDD was localized within the hepatic P100 (microsomal)fraction at all doses tested. In contrast, TCDD localized inpulmonary and renal S100 (cytosolic) fractions at all doses.The subcellular distribution of TCDD in the liver and lungsof acutely exposed B6C3F1 mice was similar to that observedin the rats. Although TCDD was concentrated within the renalP9, the remainder of TCDD in the S9 was evenly distributed betweenthe S100 and the P100 fractions of acutely exposed B6C3F1 mice.Subchronic exposure of B6C3F1 mice to 1.5 or 150 µg [³H]TCDD/kg/dayrevealed that increasing dose resulted in equal distributionof TCDD between the hepatic S9 and P9 versus concentration inthe renal P9. In addition, a dose-dependent increase in accumulationof TCDD in the hepatic P100 was accompanied by a dose-dependentincrease in TCDD localization in the renal S100 of mice subchronicallyexposed to TCDD. TCDD exposure in rats resulted in a dose-dependentincrease in the induction of CYP1A1 protein and associated enzymeactivity in hepatic, pulmonary, and renal microsomes. TCDD-inducedCYP1A2 protein levels and associated enzymatic activity wereonly present in hepatic microsomes. This is the first reportto suggest that subcellular distribution of TCDD differs betweenhepatic and nonhepatic tissues and demonstrate that the liver-specificmicrosomal localization of TCDD in female Sprague-Dawley ratsalso occurs in the liver of female B6C3F1 mice acutely or subchronicallyexposed to TCDD. In addition, these data are consistent withthe hypothesis that the hepatic sequestration of TCDD is dueto an interaction with CYP1A2. Furthermore, the lack of pulmonary/renalsequestration coupled with the lack of localization of TCDDin pulmonary/renal microsomes also supports the role of CYP1A2as a hepatic microsomal binding protein involved in TCDD sequestration.     

A dose-response study of the effects of prenatal and lactational exposure to TCDD on the immune response to influenza a virus

The goal of the current study was to evaluate the immune response to a common respiratory pathogen, influenza A virus, in mice exposed to increasing doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during development. Additionally, the treatment paradigm was designed to provide exposure throughout fetal and neonatal development, beginning on d 1 of gestation. To accomplish this, impregnated C57Bl/6 mice were treated with 0.25 microg/kg TCDD on d 0 and 7 of pregnancy, followed by 2 additional doses of 0.25, 1, or 5 microg/kg given on d 14 and postpartum d 2. The adult offspring were infected with influenza virus, and components of the adaptive and innate immune responses were evaluated. Our results show that developmental exposure to TCDD dose-responsively suppressed both the cell-mediated and antibody responses to influenza virus in female but not males. In contrast, TCDD exposure enhanced the innate immune responses in offspring of both sexes; specifically, neutrophilia and interferon (IFN) gamma levels in the lung were increased. These alterations in functional immunity did not result from overt toxicity to the immune organs, as developmental TCDD exposure did not alter the cellular composition of the thymus, spleen, or bone marrow. These findings extend our knowledge of the dose-responsive nature of immunological defects induced by developmental exposure to TCDD and offer insight regarding the dose required to alter the immune response to viral infection. Moreover, we demonstrate a clear dose at which no observable effects on immune function later in life were detected.     

A dose-response study following in utero and lactational exposure to di(2-ethylhexyl)phthalate: effects on female rat reproductive development.

Phthalates, a class of chemicals used as plasticizers, are economically important due to several industrial applications. Di(2-ethylhexyl)phthalate (DEHP) is the most commonly used phthalate plasticizer, and it has been described as a potent antiandrogen in males. We performed an extensive dose-response study following developmental exposure to DEHP and evaluated the effects on female reproductive development. Two wide ranges of doses that included dose levels relevant for human exposure as well as high doses typically used in toxicological studies were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 22. The low doses were 0.015, 0.045, 0.135, 0.405, and 1.215 mg DEHP/kg body weight (bw)/day, and the high doses were 5, 15, 45, 135, and 405 mg DEHP/kg bw/day. At the dose levels tested, no signs of maternal toxicity were observed. A significant delay in the age at vaginal opening (approximately 2 days) at 15 mg DEHP/kg bw/day and above, as well as a trend for a delay in the age at first estrus at 135 and 405 mg DEHP/kg bw/day (approximately 2 days), was observed. Liver enlargement (characteristic of phthalate exposure in rats) was limited to the 135- and 405-mg DEHP/kg bw/day doses. Anogenital distance and nipple development were unaffected. Based on the results of delayed pubertal onset, the no observed adverse effect level for female reproductive development may be set at 5 mg DEHP/kg bw/day.     

Inhibition of the hepatic cytochrome P-450-dependent monooxygenase system by rubratoxin B in male mice

The in vivo effects of rubratoxin B on hepatic mixed-function oxidase enzymes in male mice were examined. Animals were exposed to a single ip dose of 0.25, 0.5, 1.0, or 1.5 mg rubratoxin B/kg or daily doses of 0.5 mg/kg rubratoxin B for 14 days. Rubratoxin B (1 mg/kg) markedly inhibited the hepatic cytochrome P-450-dependent monooxygenase system when measured 1, 16, 48, 72, and 96 hr after a single exposure. At 48 hr, dose levels as low as 0.25 mg rubratoxin B/kg inhibited pentobarbital hydroxylase and ethylmorphine demethylase. These same enzymes, along with cytochrome P-450 content and NADPH-dependent dehydrogenase, continued to be depressed to about the same degree when animals were exposed to rubratoxin B for 14 days. NADPH-cytochrome c reductase was not affected by rubratoxin B on single or multiple exposure. These studies demonstrated that a single exposure to rubratoxin B caused a depression in activity of the cytochrome P-450 dependent monooxygenase enzyme system and that after maximum inhibition by a single dose at 48 hr, the trend was for the enzymes to return to control values.     

Cadmium-induced effects on cellular signaling pathways in the liver of transgenic estrogen reporter mice

Estrogen-like effects of cadmium (Cd) have been reported in several animal studies, and recent epidemiological findings suggest increased risk of hormone-dependent cancers after Cd exposure. The mechanisms underlying these effects are still under investigation. Our aim was to study the effects of Cd on cellular signaling pathways in vivo with special focus on estrogen signaling and to perform benchmark dose analysis on the effects. Transgenic adult ERE-luciferase male mice were exposed subcutaneously to 0.5-500 μg CdCl(2) per kg body weight (bw) or 17α-ethinylestradiol (EE2) for 3 days. These doses had no effects on organ and bw or testicular histology, indicating subtoxic exposure levels. The transgene luciferase, reporting genomic estrogen response, was significantly increased by EE2 but not by Cd. However, Cd significantly affected kinase phosphorylation and endogenous gene expression. Interestingly, gene expression changes displayed a traditional dose-response relationship, with benchmark dose levels for the expression of Mt1, Mt2, p53, c-fos, and Mdm2 being 92.9, 19.9, 7.6, 259, and 25.9 μg/kg bw, respectively, but changes in kinase phosphorylation were only detected at low exposure levels. Phosphorylation of Erk1/2 was significantly increased even in the lowest dose group, 0.5 μg/kg bw, rendering pErk1/2 a more sensitive sensor of exposure than changes in gene expression. Collectively, our data suggest that the effects triggered by Cd in vivo are markedly concentration dependent. Furthermore, we conclude that the estrogen-like effects of Cd are likely to result from a mechanism different from steroidal estrogens.     

Toxic responses to acute, subchronic, and chronic oral administrations of monochlorobenzene to rodents

Acute (single exposure), 14-d repeated exposure, 91-d subchronic, and 103-wk chronic toxicity studies of orally administered (gavage, in corn oil) monochlorobenzene were conducted in male and female Fischer-344 rats and B6C3F1 hybrid mice. A single exposure to 4000 mg/kg was lethal to male and female rats, while a single exposure to a dose as low as 1000 mg/kg was lethal to mice. Fourteen daily exposures to 1000 mg/kg caused death in rats of both sexes, but neither survival nor clinical health were compromised at 500 mg/kg in rats or mice. In the 91-d studies, wherein monochlorobenzene was administered once daily, 5 d/wk, survival was reduced by doses of 500 mg/kg and higher in rats, and by doses of 250 mg/kg and higher in mice. Dose-dependent necrosis of the liver (hepatocytes), degeneration or focal necrosis of the renal proximal tubules, and lymphoid or myeloid depletion of the spleen, bone marrow, and thymus (mild to severe) were produced by doses of 250 mg/kg or greater of monochlorobenzene in both sexes of rats and mice, although the incidences of these lesions varied considerably by sex and species. Consistent changes in the circulating blood components were not observed, but a mild porphyrinuria was detected at the higher doses. No toxic effects were observed at doses of 125 mg/kg or less. In the 2-yr studies, wherein monochlorobenzene was administered once daily, 5 d/wk, doses of 30 or 60 mg/kg in male mice and 60 or 120 mg/kg in female mice and male and female rats did not produce any evidence of toxicity. Doses of 60 or 120 mg/kg caused slight (statistically significant at 120 mg/kg; p less than 0.05) increases in the frequencies of male rats with neoplastic nodules of the liver. Increased tumor frequencies were not observed in female rats or in male or female mice receiving monochlorobenzene.     

Dose-dependent localization of TCDD in isolated centrilobular and periportal hepatocytes

Dose-response relationships for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suggest a differential sensitivity of liver cell types to the induction of cytochrome P450 gene expression, and that the induction of hepatic protein CYP1A2 causes sequestration of TCDD. In addition, immunolocalization of hepatic CYP1A1/1B1/1A2 proteins is not uniform after exposure to TCDD. The mechanism for the regio-specific induction of hepatic P450s by TCDD is unknown, but may involve the differential distribution of participants in the AhR-mediated pathway and/or regional P450 isozymes, as well as, non-uniform distribution/sequestration of TCDD. Therefore, this study examined the effects of TCDD in unfractionated, centrilobular and periportal hepatocytes isolated from female Sprague-Dawley rats acutely exposed (3 days) to a single oral dose of 0.01-10.0 microg [3H]TCDD/kg. A dose- dependent increase in concentration of TCDD was accompanied by a dose- dependent increase in CYP1A1, CYP1A2, and CYP1B1 mRNA expression and associated enzymes in all liver-cell populations. Centrilobular hepatocytes showed a 2.7- to 4.5-fold higher concentration of TCDD as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Centrilobular hepatocytes also exhibited an elevated MROD activity as compared to the periportal hepatocytes at doses up to 0.3 microg TCDD/kg. Furthermore, centrilobular hepatocytes showed an elevated concentration of induced CYP1A2 and CYP1B1 mRNA as compared to periportal hepatocytes within the 0.01- and 0.3-microg TCDD/kg- treatment groups. This is the first study to demonstrate that a dose- dependent difference in distribution of TCDD exists between centrilobular and periportal cells that might be related to regional differences in P450 induction.      

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice

Three-month-old male $\text{C57B1}\text{6}$ mice were given single oral doses of 0, 100, 150, or 200 μg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg. The LD50 was 114 μ/kgg. In mice that died, depletion of the thymus and spleen were consistently found and edema and terminal hemorrhages occurred frequently. In a second experiment, 4-month-old male mice were dosed po with 0, 0.2, 1.0, 5.0 or 25 μg/kg, once a week for 2 or 6 weeks. Some deaths and growth retardation occurred in the 25 μg/kg dose group. Significantly increased liver and decreased thymus weights were found in the 1, 5 and 25 μg/kg dose groups. Total neutrophils were increased significantly, whereas hemoglobin values and mean corpuscular hemoglobin concentrations were decreased significantly after 6 doses of 25 μg/kg. Total serum protein and α-, β-, and γ-globulins were significantly decreased. TCDD was porphyrogenic. The hepatic porphyria was probably associated with liver damage. Degenerative and necrotic changes in the liver were essentially centrilobular and were accompanied by cellular infiltrates and ceroid pigment deposition. Proliferation of bile duct and bile duct epithelial cells occurred. Lipid accumulation was centrilobulary localized in the mice receiving 0.2 μg/kg, was more pronounced in the mice of the intermediate dose levels, and involved hepatocytes throughout the lobule in the 25 μg/kg dose group.