Effect of the environmental pollutant hexachlorobenzene (HCB) on the neuronal differentiation of mouse embryonic stem cells.

Exposure to persistent environmental pollutants may constitute an important factor on the onset of a number of neurological disorders such as autism, Parkinson's disease, and Attention Deficit Disorder (ADD), which have also been linked to reduced GABAergic neuronal function. GABAergic neurons produce γ-aminobutyric acid (GABA), which is the main inhibitory neurotransmitter in the brain. However, the lack of appropriate models has hindered the study of suspected environmental pollutants on GABAergic function. In this work, we have examined the effect of hexachlorobenzene (HCB), a persistent and bioaccumulative environmental pollutant, on the function and morphology of GABAergic neurons generated in vitro from mouse embryonic stem (ES) cells. We observed that: (1) treatment with 0.5 nM HCB did not affect cell viability, but affected the neuronal differentiation of ES cells; (2) HCB induced the production of reactive oxygen species (ROS); and (3) HCB repressed neurite outgrowth in GABAergic neurons, but this effect was reversed by the ROS scavenger N-acetylcysteine (NAC). Our study also revealed that HCB did not significantly interfere with the function of K+ ion channels in the neuronal soma, which indicates that this pollutant does not affect the maturation of the GABAergic neuronal soma. Our results suggest a mechanism by which environmental pollutants interfere with normal GABAergic neuronal function and may promote the onset of a number of neurological disorders such as autism and ADD.

Further analysis of behavioral and endocrine consequences of chronic exposure of male Wistar rats to subtoxic doses of endocrine disruptor chlorobenzenes.

Many chemicals utilized by humans are present as environmental pollutants and may influence homeostasis from neurological, immunological, endocrinological and/or behavioral aspects. Such agents, acting alone or in ambient mixtures, may be biologically active even at extremely low doses, and it may be postulated that stable, bioaccumulative, reactive endocrine disruptors may affect central and/or peripheral secretion of arginine-vasopressin (AVP) and oxytocin (OXT) and thereby related physiological and behavioral functions, potentially leading to disorders in exposed subjects. The primary aim of this study was to demonstrate effects of chronic exposure to a low dose of an orally administered chlorobenzene mixture on anxiety-related and aggressive behavior mediated largely by AVP and OXT. Chlorobenzenes were applied to model ambient mixtures of endocrine disruptors. Adult, male Wistar rats were exposed daily to 0.1 µg/kg of 1,2,4-trichlorobenzene and hexachlorobenzene via a stomach tube for 30, 60 or 90 days, after which anxiety-related and aggressive behavioral elements were examined in open-field, elevated plus maze and resident-intruder tests. The plasma levels of AVP, OXT and adrenocorticotrophic hormone at the endpoints were measured by radioimmunoassay or immunochemiluminescence assay. The levels of basal and serotonin- or norepinephrine-stimulated AVP and OXT secretion in pituicyte cultures prepared from the posterior lobe of the pituitaries were also measured. The hormone levels proved to be increased to extents depending on the duration of exposure to the chlorobenzenes. Several anxiety-related and aggressive behavioral elements were also enhanced following chlorobenzene exposure, while certain explorative and locomotive elements of the animals were decreased. As both physiological and behavioral elements were modulated by chronic, subtoxic doses of chlorobenzenes, it is concluded that doses of such environmental pollutants low enough to fall outside the range of legal regulation may pose potential risks of anxiogenic and/or aggressive consequences in exposed subjects, including humans.

Effects of aging and sediment composition on hexachlorobenzene desorption resistance compared to oral bioavailability in rats.

Studies were conducted to assess the effects of black carbon, clay type and aging (1-1.5yr) on desorption and bioavailability of hexachlorobenzene (HCB) in spiked artificial sediments. Tenax (a super sorbent)-mediated desorption was used to examine the effects of these parameters on the physicochemical availability of HCB. The Tenax-mediated desorption of HCB from the four aged artificial sediments exhibited biphasic kinetics. The fast desorbing fractions ranged from 64.8% to 22.3%, showing reductions of 4.0-18.9% compared with freshly-spiked sediments. Statistical analysis on the fast desorbing fractions showed that all three treatment effects (i.e., montmorillonite clay, black carbon content, and aging) were significant. Two sediments with higher black carbon content exhibited much greater aging effects (i.e., greater reduction in fast desorbing fraction) than the other two sediments without the addition of black carbon. For both freshly-spiked and aged sediments, the desorption resistant sediment-bound HCB (i.e., slow desorbing fraction) correlated reasonably well to previously reported rat fecal elimination of HCB, which is a measure of the non-bioavailable fraction of sediment-bound HCB. A similar correlation was also observed between fast desorbing fraction and previously reported accumulation of HCB in the rat body (carcass+skin). These observations suggest that physicochemical availability, as defined by the desorption of HCB from sediments, provides a reasonable prediction of the oral bioavailability of sediment-bound HCB to rats. These results showed that montmorillonite clay, black carbon and aging reduced physicochemical availability and ultimately bioavailability of sediment-bound HCB.

Cytotoxic effects induced by hexachlorobenzene in Squilla mantis (L.) (Crustacea, Stomatopoda).

Contamination of marine environments by hexachlorobenzene (HCB) represents a serious concern for potential consequences on ecosystem and human health. Despite this, information on cytotoxic effects on marine organisms is still largely lacking. In this study, we investigated cytotoxic effects induced by HCB on gonads and muscular tissue of Squilla mantis by analysing Na(+)/K(+)-ATPase activity and plasma membrane fluidity. This crustacean species was selected as a model for its habitat, trophic level, feeding behavior, and commercial exploitation for human consumption. Time course experiments revealed that low concentrations of HCB (i.e. 50 nM) determine an exponentially decrease of Na(+)/K(+)-ATPase activity and a significant modification of cellular membrane fluidity. Significant negative relationships between Na(+)/K(+)-ATPase activity and membrane fluidity were observed, suggesting that changes in the structure and packing of cellular membranes induced by HCB may be the primary factor affecting the activity of essential bilayer-associated enzymes. Overall these findings suggest that even small concentrations of HCB may determine important changes on cell metabolism with potential cascade effects on recruitment of this commercial species.

Quantitative analysis of liver GST-P foci promoted by a chemical mixture of hexachlorobenzene and PCB 126: implication of size-dependent cellular growth kinetics.

The objectives of this study were twofold: (1) evaluating the carcinogenic potential of the mixture of two persistent environmental pollutants, hexachlorobenzene (HCB) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126), in an initiation-promotion bioassay involving the development of pi glutathione S-transferase (GST-P) liver foci, and (2) analyzing the GST-P foci data using a biologically-based computer model (i.e., clonal growth model) with an emphasis on the effect of focal size on the growth kinetics of initiated cells. The 8-week bioassay involved a series of treatments of initiator, two-thirds partial hepatectomy, and daily oral gavage of the mixture of two doses in male F344 rats. The mixture treatment significantly increased liver GST-P foci development, indicating carcinogenic potential of this mixture. Our clonal growth model was developed to simulate the appearance and development of initiated GST-P cells in the liver over time. In the model, the initiated cells were partitioned into two subpopulations with the same division rate but different death rates. Each subpopulation was further categorized into single cells, mini- (2-11 cells), medium- (12-399 cells), and large-foci (>399 cells) with different growth kinetics. Our modeling suggested that the growth of GST-P foci is size-dependent; in general, the larger the foci, the higher the rate constants of division and death. In addition, the modeling implied that the two doses promoted foci development in different manners even though the experimental foci data appeared to be similar between the two doses. This study further illustrated how clonal growth modeling may facilitate our understanding in chemical carcinogenic process.

Effect of organic carbon content, clay type, and aging on the oral bioavailability of hexachlorobenzene in rats.

Bioavailability of lipophilic chemicals is influenced by the physicochemical properties of soils/sediment such as particle size, pH, clay, and organic carbon content. The present study investigated the effects of sediment composition and aging on the oral bioavailability of hexachlorobenzene (HCB) in rats. Formulated sediments were prepared using various ratios of kaolinite and montmorillonite clay, sand, peat moss, and black carbon, spiked with (14)C-HCB, and orally administered to rats prior to and after one year of aging in dark at 10 degrees C. In the nonaged sediments there was a 21 to 45% reduction in the oral bioavailability of HCB when compared to the corn oil standard without any clear pattern of the impact of the sediment clay and/or organic carbon content. One year of aging resulted in statistically significant (p = 0.049) reduction in the oral bioavailability of HCB from the sediments compared to the corn oil standard and nonaged sediment indicating stronger interactions between HCB and sediment contents with aging. The mean reduction in oral bioavailability after one year of aging ranged from approximately 5 to 14% greater than that observed for nonaged sediments. The fecal elimination of the HCB-derived radioactivity from the one-year-aged sediments was much higher than the nonaged sediments, consistent with the lower absorption from the gastrointestinal tract due to lower desorption of HCB from the aged sediments. Increase in the fecal elimination and decrease in oral bioavailability of (14)C-HCB was related to the increase in clay and black carbon.

Hepatic arachidonic acid metabolism is disrupted after hexachlorobenzene treatment.

Hexaclorobenzene (HCB), one of the most persistent environmental pollutants, can cause a wide range of toxic effects including cancer in animals, and hepatotoxicity and porphyria both in humans and animals. In the present study, liver microsomal cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolism, hepatic PGE production, and cytosolic phospholipase A2 (cPLA2) activity were investigated in an experimental model of porphyria cutanea tarda induced by HCB. Female Wistar rats were treated with a single daily dose of HCB (100 mg kg(-1) body weight) for 5 days and were sacrificed 3, 10, 17, and 52 days after the last dose. HCB treatment induced the accumulation of hepatic porphyrins from day 17 and increased the activities of liver ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and aminopyrine N-demethylase (APND) from day 3 after the last dose. Liver microsomes from control and HCB-treated rats generated, in the presence of NADPH, hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), 11,12-Di HETE, and omega-OH/omega-1-OH AA. HCB treatment caused an increase in total NADPH CYP-dependent AA metabolism, with a higher response at 3 days after the last HCB dose than at the other time points studied. In addition, HCB treatment markedly enhanced PGE production and release in liver slices. This HCB effect was time dependent and reached its highest level after 10 days. At this time cPLA2 activity was shown to be increased. Unexpectedly, HCB produced a significant decrease in cPLA2 activity on the 17th and 52nd day. Our results demonstrated for the first time that HCB induces both the cyclooxygenase and CYP-dependent AA metabolism. The effects of HCB on AA metabolism were previous to the onset of a marked porphyria and might contribute to different aspects of HCB-induced liver toxicity such as alterations of membrane fluidity and membrane-bound protein function. Observations also suggested that a possible role of cPLA2 in the early increase of AA metabolism cannot be excluded. However, the existence of other pathway(s) for metabolizable AA generation different from cPLA2 activation is also proposed.

The role of type I and type II 5' deiodinases on hexachlorobenzene-induced alteration of the hormonal thyroid status.

Treatment of male Wistar rats with hexachlorobenzene (HCB) (1000 mg/kg b.w.) for 3-30 days decreases circulating levels of thyroxine (T4) but does not affect triiodothyronine (T3). Time courses were determined for 5' deiodinase type I (5' D-I) activity in thyroid, liver, and kidney and 5' deiodinase type II (5' D-II) activity in brown adipose tissue (BAT) to test the possibility that increased deiodinase activity might contribute to the maintenance of the serum T3 level. Specific 5' D-I activity was increased in the thyroid at 21 days and thereafter. No significant changes were observed in the liver, however, total 5' D-I activity in this tissue was increased at 30 days of treatment as a consequence of liver weight enhancement. HCB decreased kidney 5' D-I activity after 15 days, and BAT 5' D-II activity after 21 days of treatment. Total body 5' D-I activity was significantly increased by 30 days of HCB-treatment. HCB increased the activity of hepatic T4 uridine diphosphoglucuronosyl transferase (UDPGT) in a time-dependent manner, without changes in T3 UDPGT. We propose that increased T4 to T3 conversion in the thyroid and in the greatly enlarged liver may account for the maintenance of serum T3 concentration in hypothyroxinemic HCB-treated rats.

Toxicogenomics of subchronic hexachlorobenzene exposure in Brown Norway rats.

Hexachlorobenzene (HCB) is a persistent environmental pollutant with toxic effects in man and rat. Reported adverse effects are hepatic porphyria, neurotoxicity, and adverse effects on the reproductive and immune system. To obtain more insight into HCB-induced mechanisms of toxicity, we studied gene expression levels using DNA microarrays. For 4 weeks, Brown Norway rats were fed a diet supplemented with 0, 150, or 450 mg HCB/kg. Spleen, mesenteric lymph nodes (MLN), thymus, blood, liver, and kidney were collected and analyzed using the Affymetrix rat RGU-34A GeneChip microarray. Most significant (p < 0.001) changes, compared to the control group, occurred in spleen, followed by liver, kidney, blood, and MLN, but only a few genes were affected in thymus. This was to be expected, as the thymus is not a target organ of HCB. Transcriptome profiles confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria, and the reproductive system. In line with previous histopathological findings were increased transcript levels of markers for granulocytes and macrophages. New findings include the upregulation of genes encoding proinflammatory cytokines, antioxidants, acute phase proteins, mast cell markers, complements, chemokines, and cell adhesion molecules. Generally, gene expression data provide evidence that HCB induces a systemic inflammatory response, accompanied by oxidative stress and an acute phase response. In conclusion, this study confirms previously observed (immuno)toxicological effects of HCB but also reveals several new and mechanistically relevant gene products. Thus, transcriptome profiles can be used as markers for several of the processes that occur after HCB exposure.

Hexachlorobenzene, a dioxin-like compound, disrupts auditory function in rat.

Hexachlorobenzene (HCB) is a dioxin-like compound widely distributed in the environment. In this study, we investigated the effects of HCB on the cochlea. Conscious free-moving rats were given HCB per os daily for 4 weeks at doses of 0.16, 4 or 16 mg/kg in olive oil, whereas the control group received olive oil only. The effects of HCB were evaluated at various time intervals, by measuring auditory nerve acoustic thresholds and plasma thyroid hormone concentration by radioimmunoassay. Histological evaluation involved surface preparation and scanning electron microscopy observations of cochlear hair cells. At a dose of 0.16 mg/kg, HCB induced no loss of acoustic sensitivity, whereas at 4 mg/kg, it induced cochlear sensitivity deficits at the mid-frequencies (2-16 kHz) with complete recovery once treatment was stopped. At a dose of 16 mg/kg, permanent threshold shifts were observed at all frequencies tested (from 1 to 32 kHz). Morphological studies showed no cochlear hair cell loss or alteration of stereocilia. HCB treatment reduced circulating thyroxine concentrations. Thyroidectomy had no effect on cochlear sensitivity in control animals. Thus, HCB is a potent oto-toxicant, and its ototoxicity may be independent of its thyroidal effects.

Hexachlorobenzene-induced incisor degeneration in Sprague-Dawley rats.

Hexachlorobenzene (HCB) was administered to female Sprague-Dawley rats in a 13-week toxicity study conducted for the National Toxicology Program (NTP). Groups of 10 rats were treated by gavage with 0.0, 0.03, 0.1, 0.3, 1.0, 3.0, 10, or 25 mg/kg HCB in corn oil 5 days/week for 13 weeks. Dose-dependent, region-specific, maxillary incisor degeneration was observed in animals given HCB at doses of 1.0 mg/kg/day and higher. Treatment with 1.0 mg/kg/day resulted in focal mesenchymal cell vacuolation and focal osteodentin formation within the pulp. Lesions were restricted to a short linear region subjacent to the odontoblast layer along the lateral aspect of the incisor at the level of the dentin-enamel interface. When observed in both incisors, lesions were bilaterally symmetric. Treatment with 3.0 mg/kg/day resulted in lateral and medial pulp lesions and focal odontoblast degeneration with dentin niche formation along the lateral incisor margin in some animals. Treatment with 10 mg/kg/day resulted in a higher incidence of lateral dentin niches and a low incidence of medial dentin niches. In animals treated with 25 mg/kg/day, a high incidence of bilaterally symmetric, lateral and medial, dentin niches were observed. The morphology and distribution of HCB-induced incisor lesions in rats are consistent with injury to a selective population of preodontoblasts.

Modeling ingestion as an exposure route for organic chemicals in earthworms (Oligochaeta).

Earthworms take up chemicals from both soil pore water and food, but the quantitative contribution of each route is unclear. In this paper, a model is presented for the accumulation of organic chemicals in earthworms, including a compartment for the gut contents. A Monte Carlo screening method is used to calibrate the model simultaneously to four experimental data sets for hexachlorobenzene (HCB) in the compost worm (Eisenia andrei). The calibration procedure shows that the dominant route of exposure is across the gut wall. Nevertheless, predicted body residues of HCB are generally no more than 20% higher than the estimate based on equilibrium partitioning (EP), independent of the uncertainties in all of the model parameters. The deviation from EP is mainly related to the digestive efficiency of the earthworm. Body residues are not expected to exceed the EP estimate by more than 50% for other chemicals or other earthworm species. The model is particularly useful when the organism's food source is specifically contaminated and EP cannot be applied.

Hexachlorobenzene-induced early changes in ornithine decarboxylase and protein tyrosine kinase activities, polyamines and c-Myc, c-Fos and c-Jun proto-oncogenes in rat liver.

Hexachlorobenzene (HCB) is a lipophilic chemical compound that is widely distributed in the environment. HCB is known to cause liver tumors in experimental animals. In the present study the in vivo effect of HCB treatment on ornithine decarboxylase (ODC) and protein tyrosine kinase (PTK) activities, free polyamine content, and c-Myc, c-Fos, and c-Jun protein levels in rat liver were investigated. HCB (1000 mg/kg body weight) increased hepatic immunodetectable c-Myc, c-Fos, and c-Jun levels after 6 h, and ODC activity and spermine and putrescine content after 18 and 24 h, while maximum stimulation of PTK activity occurred at 12 h. PTK and ODC activities varied in a dose-dependent manner. The time-course of c-Myc, c-Fos, and c-Jun protein levels was different for each proto-oncogene. They were all elevated at the second day of treatment, while only c-Fos and c-Jun remained elevated after 10 days of HCB exposure. These data jointly suggest that the increase in ODC activity may be the consequence of proto-oncogene induction. The alterations in PTK activity suggest that the growth factor signal transduction pathway may be involved in the regulation of the proto-oncogene levels or/and ODC activity. The decrease in PTK activity after the first day, even in the presence of alpha-D-Difluoromethylornithine (DFMO), an inhibitor of ODC activity, suggests that it is not regulated by polyamines. These results may be relevant to the early molecular events involved in HCB tumor promoter activity in rat liver.

High-fat diet enhances the accumulation of hexachlorobenzene (HCB) by pregnant rats during continuous exposure to HCB.

To investigate the influence of a high-fat diet on HCB distribution and accumulation, pregnant rats in study 1 were fed a high-fat or control diet containing HCB, and, in study 2, pregnant rats were given a single HCB dose by intragastric gavage and HCB-free high-fat or control diet. In study 1, the high-fat diet group had higher HCB concentrations in fat tissues and liver than did the controls. In study 2, although the total amounts of HCB in the fat tissue and liver were greater in the high-fat diet group than in the controls, no significant differences in HCB concentration were observed between the two groups. The high-fat diet group also showed more fecal excretion of HCB. Therefore, HCB accumulation in rats fed a high-fat diet was enhanced more by continuous exposure to HCB than by administration of a single dose.

Two immunotoxicity ring studies according to OECD TG 407-comparison of data on cyclosporin A and hexachlorobenzene.

Two international ring studies were performed to develop appropriate parameters within standard toxicology study for screening of immunotoxicological potential of unknown substances. These studies followed OECD TG 407 and included a number of additional examinations. CSA was selected as model for its immunosuppressive and HCB as model for its immunostimulating effects. Reproducibility of data was defined by significant findings in at least 50% of participating laboratories. In-life clinical observations, values for WBC parameters, and changes of lymphoid organ weights suggested immune effects. Elevated IgM titers indicated increased antibody formation in HCB-exposed rats. Cellularity of T-cell compartments in thymus (medulla), spleen (PALS), and lymph nodes (paracortical zone of mesenteric and popliteal LN) were dose dependently decreased in CSA-treated rats. The numbers of follicular germinal centers were reduced in LN. HCB induced cellular proliferation in spleen marginal zones and endothelial activation in HEV of mesenteric and popliteal LN and GALT and in small pulmonary venules. Data obtained by specific immune parameters indicated immune effects; however, statistical inference was limited to low numbers of participating laboratories. In spleen, both substances decreased lymphoblast proliferation after ConA mitogen stimulation. Reduced numbers of antibody-forming cells in PFC assay indicated impaired T-cell-dependent humoral immunity by CSA, which was not seen for HCB. Altered fractions for B- and T-cell subpopulations were identified in spleen for both substances. In order to predict immunomodulatory effects of CSA or HCB, histomorphologic examination of lymphoid tissues resulted in the most reliable and sensitive data to distinguish immunosuppression and -stimulation.

Respuesta hepática en ratas tratadas con hexaclorobenceno utilizando diferentes fuentes de carbohidratos en la dieta / Hepatic response in rats treated with hexachlorobenzene fed differing in the carbohydrate source

Ratas Wistar machos de peso 280 g, recibieron dosis total de dos gramos de hexaclorobenceno (HCB), administrado en lña dieta semipurificada normoproteica isocalórica (18 por ciento de caseína, 68,5 por ciento de hidrato de carbono, que contenía almidón o sacarosa), durante 8 semanas. La capacidad del HCB de estimular la actividad del sistemamicrosómico hepático fue evaluada in vivo mediante el test de parálisis de las patas traseras utilizando zoxazolamina (i.p.). Fueron evaluados el peso del hñígado y su composición bioquímica en proteínas, lípidos, ADN y ARN. Las muestras de hígado fueron estudiadas histologicamente. El tratamiento con HCB no modificó el patrón de ingestión de alimentos en ambos grups tratados con sacarosa o almidón. Los animales con sacarosa mostraronb menor ingestión de alimento. Sin embargo, el desarrollo y el peso corporal fueron semejantes en ambos grupos al final del experimento. Los animales tratados con HCB presentaron, independientemente de la fuente de carbohidratos de la dieta, reducción del tiempo de parálisis de las patas traseras por la zoxazolamina, aumento de peso del hígado y de los contenidos de proteínas y lípidos, junto a una menor concentración hepática de ADN, Histológicamente se observó hipertrofia de hepatocitos centrolobulillares y cúmulo graso en hepatocitos de la zona periportal. los resultados de este estudio mostraron que, bajo los parámetros estudiados, las ratas expuestas al HCB presentaron inducción del sistema microsómico hepático. Esta inducción fue acompañada por un agrandamiento de hepatocitos centrolobulillares y aumento de peso del hígado y de su contenido en proteínas. (AU)

Heme metabolism after discontinued hexachlorobenzene administration in rats: possible irreversible changes and biomarker for hexachlorobenzene persistence.

The aim of the present study was to determine whether short-term administration of hexachlorobenzene (HCB) (1 g/kg body wt., suspended in water, 5 days/week), could cause and maintain marked porphyria in the absence of the exogenous drug, and whether porphyria parameters can be useful as biomarkers of HCB persistence in rats. Hepatic uroporphyrinogen decarboxylase activity, its inhibitor formation, porphyrin content and composition were studied in Wistar rats treated with the fungicide for 1, 2, 3, or 4 weeks and then withdrawn for a 20-week period. The time course of urinary porphyrin excretion was studied for 7 weeks either by continuous treatment for the entire period, or a 1-week HCB administration. The degree of porphyria achieved by rats after 20 weeks of suspended HCB administration was severe, independent of the length of the treatment, and even higher than that observed in animals analysed immediately at the end of each treatment. Rats treated with HCB for 1 week showed a modest decrease in uroporphyrinogen decarboxylase and low inhibitor formation, and exhibited a greater enzyme inhibition, inhibitor formation, hepatic porphyrin accumulation, and an altered pattern of porphyrin composition in the absence of the exogenous drug. Independent of the treatment, urinary porphyrins rose after a delay of 5 weeks. Substantial amounts of HCB were still found in fat of rats treated with HCB for 1 week, after a withdrawal period of 20 weeks. These results suggest that the high persistence of HCB in tissues acts as a continuous source of the xenobiotic, and stimulus for heme biosynthesis derangement. The alterations induced by HCB within 1 week of treatment could be regarded as an initial trigger for irreversible damage on heme metabolism. Thus, abnormalities in heme biosynthesis can be considered effective markers of HCB persistence in rats or of irreversible HCB-induced damage. Taking into account the delayed and enhanced metabolic effects of HCB, it is advisable that porphyria parameters should be evaluated not only immediately after exposure, but also some time afterwards, especially in susceptible and occupationally-exposed populations.

Dosing via gavage or diet for reproduction studies: a pilot study using two fat-soluble compounds-hexachlorobenzene and aroclor 1254.

The choice of a dosing route for in vivo toxicological tests is often dictated by practical constraints. Reproduction studies are particularly challenging in this regard since the determination of no-effect levels and allowable daily intakes from reproduction data encompasses exposure of the dam to the test xenobiotic prior to pregnancy, during gestation and during lactation. The fetus/infant can be exposed to the xenobiotic as well as the dam's metabolic products of the test xenobiotic during gestation and lactation. We initiated a series of two-litter, pilot reproduction studies with Sprague-Dawley and Fischer 344 rats to specifically ascertain the amount of xenobiotic and its metabolites ingested by the nursing neonate on lactation days 4, 7, 12, 17 and 21, when its dam received the xenobiotic via its diet or by gavage. The xenobiotics studied in this initial series of experiments were hexachlorobenzene (HCB) and Aroclor(R) 1254 (polychlorinated biphenyls; PCBs). The dams were dosed for 28 days, mated to untreated males and then remated approximately 2 weeks after weaning their first litter to a second untreated male. Dietary levels of 10 ppm HCB or 10 ppm PCBs, and gavage doses of 0.9 mg HCB or 0.8 mg PCBs/kg body weight/day were chosen and resulted in similar doses of HCB and PCBs per unit of the body weight of the dam during the premating period. There were no apparent toxicological effects regarding the dam nor were any of the reproduction parameters (feed consumption, dam weight, litter size, pup weight, external anomalies and day 4 viability index) significantly different from control values. Following impregnation, the body weight of the dam increased appreciably during gestation, but its feed consumption increased only slightly. During lactation, the dam's feed consumption increased markedly while its body weight increased slightly. Consequently, when dams received the xenobiotic in their diet they consumed slightly less xenobiotic per unit of body weight during gestation when compared to the gavaged dams, whereas the situation was dramatically reversed during lactation. While the greater consumption of xenobiotic by the dietary-dosed dams during lactation did result in more HCB (P

An approach for modeling noncancer dose responses with an emphasis on uncertainty.

This paper presents an approach for characterizing the probability of adverse effects occurring in a population exposed to dose rates in excess of the Reference Dose (RfD). The approach uses a linear threshold (hockey stick) model of response and is based on the current system of uncertainty factors used in setting RfDs. The approach requires generally available toxicological estimates such as No-Observed-Adverse-Effect Levels (NOAELs) or Benchmark Doses and doses at which adverse effects are observed in 50% of the test animals (ED50s). In this approach, Monte Carlo analysis is used to characterize the uncertainty in the dose response slope based on the range and magnitude of the key sources of uncertainty in setting protective doses. The method does not require information on the shape of the dose response curve for specific chemicals, but is amenable to the inclusion of such data. The approach is applied to four compounds to produce estimates of response rates for dose rates greater than the RfD.

Studies on the mechanism of uroporphyrinogen decarboxylase inhibition in hexachlorobenzene-induced porphyria in the female rat.

Hexachlorobenzene (HCB)-induced porphyria occurs in female, but not male, rats after a delay of 35 days following HCB treatment. Uroporphyrinogen decarboxylase (UROD) inhibition has been proposed as a primary causative event. To determine whether there also exists a delay phase and a sexual dimorphism for UROD inhibition, groups of male and female rats were given HCB (100 mg/kg/day) from Days 1 to 5. Hepatic uroporphyrin III was markedly increased only after Day 33. Liver cytosol UROD activity in HCB-treated female rats with porphyria at Days 33, 40, 47, 54, and 100 was decreased by over 70% compared to concurrent control, whereas treated male rats as well as nonporphyric female rats had UROD activity comparable to control levels at Days 6, 12, 19, 26, 33, 40, 47, and 54. Level of immunoreactive UROD in cytosol of porphyric rats was not modified by HCB. No gender-related differences in liver cytosol radiolabel level ([14C]HCB given as the fifth dose) were found at Days 6 and 30. Chromatography of liver cytosol showed nonspecific binding of radiolabel to proteins for males, porphyric and nonporphyric females, and loss of UROD activity did not correlate with the amount of radiolabel in the UROD-containing fractions. Thus, the gender-specific decrease in UROD activity observed when porphyria develops in female rats (delay of about 4 weeks), as well as the persistence of low activity and porphyria for months, suggests that UROD inhibition was causally related to porphyria.