Biomedical subjects

T A Gasiewicz

Publications and source records attributed to T A Gasiewicz.

At least 73 records · Page 4Linked to original sources

PubMed1989

Selective inhibition of polymorphonuclear neutrophil activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Although the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), via its interaction with the Ah receptor, is an extremely potent carcinogen and immunosuppressive agent in experimental animals, its possible actions on polymorphonuclear (PMN) function have not been determined. In addition to their importance against infectious organisms, PMNs have been implicated in antitumor resistance. The present studies examined the effects of in vivo exposure to TCDD on PMN function in B6C3F1 (TCDD sensitive, presence of high affinity Ah receptor) and DBA/2N (TCDD resistant at low doses, defective Ah receptor) mice. Animals received a single oral exposure of 5 or 10 micrograms/kg of TCDD and PMNs were obtained 5 days later from the peritoneal cavity following elicitation with sodium caseinate. TCDD reduced the cytolytic and cytostatic activity of PMA-activated PMNs in B6C3F1, but not in DBA/2N mice, suggesting that this response segregates with the Ah locus. Furthermore, TCDD was found to bind specifically to PMNs from Ah-responsive mice. Neither the production of superoxide and hydrogen peroxide nor degranulation, the latter measured by beta-glucuronidase release, was impaired. Supernatants recovered from PMN cell cultures of TCDD-sensitive mice, but not from resistant DBA/2N mice, showed reduced killing capacity for actinomycin D-treated L929 tumor cells, while their ability to bind to tumor cells was not altered. These data suggest that TCDD interferes with PMN-mediated tumor cell killing by altering the production or secretion of a cytolytic factor. Examination of bone marrow stem cells revealed that granulocytic but not monocytic colonies were reduced after TCDD exposure in vivo and in vitro. Although mature PMNs had detectable levels of Ah receptor, exposure in vitro of these cells to TCDD had no effect on antitumor activity. Thus, it is possible that TCDD may affect PMNs at the level of hematopoiesis, via a direct interaction with granulocyte precursor cells, or modulate PMNs at different stages of maturation.

Animals↗

PubMed1989

Lymphocyte stem cell alterations following perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Perinatal exposure of experimental animals to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to thymic atrophy and a suppression of cell-mediated immunity that is more severe and persistent than that caused by adult exposure, suggesting that events involved in the maturation of the immune system are particularly sensitive to TCDD. We report here that perinatal TCDD exposure produces an alteration in the lymphocyte stem cell population in the fetus and neonate, as evidenced by a significant reduction in the lymphocyte stem cell-specific enzyme terminal deoxynucleotidyl transferase (TdT). After maternal treatment with a single dose of TCDD (10 micrograms/kg of body weight) on gestational day (gd) 14, TdT biosynthesis and TdT-specific mRNA were reduced more than 50% in fetal liver lymphoid cells on gd 18. An even more extensive reduction was seen in neonatal bone marrow through postnatal day 18. In contrast, thymic TdT synthesis appeared to be relatively unaffected on a per cell basis by perinatal TCDD exposure, although the actual number of TdT-synthesizing thymocytes was diminished due to extensive thymic atrophy. These effects occurred at concentrations of 1-31 fg of TCDD/mg of thymus. Flow cytometric analysis of thymocyte surface marker expression revealed a slight decrease in the percentage of Lyt-2+L3T4+ thymocytes on gd 18 and postnatal day 4. This alteration was no longer apparent by postnatal day 11, when marrow TdT biosynthesis was most suppressed. These results suggest that TCDD-induced thymic atrophy during the perinatal period may be due, in part, to an effect on the prothymocyte.

Animals↗

PubMed1988

In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster.

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.

Animals↗

PubMed1988

The role of the Ah locus in hexachlorobenzene-induced porphyria. Studies in congenic C57BL/6J mice.

The role of the Ah locus in hexachlorobenzene (HCB)-induced porphyria and the possible involvement of P-450 cytochromes P(1)450 and P(3)450 in the pathogenesis of this disease were investigated in two congenic strains of C57BL/6J mice that differ only at this locus. Female B6-Ahb mice (Ah receptor: approximately 30-70 fmol/mg of cytosolic protein) and B6-Ahd mice (Ah receptor: undetectable) were pretreated with iron (500 mg/kg) and then fed a diet containing 0 or 200 p.p.m. of HCB for up to 17 weeks. Mice from the two strains consumed similar amounts of HCB. Urinary excretion of porphyrins was increased after 7 weeks of HCB treatment in B6-Ahb mice, and after 15 weeks was over 200 times greater than that of mice given iron only. In B6-Ahd mice, porphyrin excretion did not begin to increase until after 13 weeks, and after 15 weeks was only six times greater than that of controls. Similar differences were seen in the 15-week hepatic porphyrin concentrations (B6-Ahb: 1110 +/- 393; B6-Ahd: 17.6 +/- 14.5; controls: approximately 0.20 nmol/g). Uroporphyrinogen decarboxylase (EC 4.1.1.37) activity was diminished by 70 and 20% in B6-Ahb B6-Ahd mice respectively after 15 weeks of treatment with HCB. Cytochromes P(1)450 and P(3)450 were measured in hepatic microsomes (microsomal fractions) by radioimmunoassay and immunoblotting, using antisera raised against the orthologous rat isoenzymes P450c and P450d. HCB induced small amounts of a protein recognized by anti-P450c (P(1)450) in B6-Ahd mice, but not in B6-Ahd mice. Relatively large amounts of a protein recognized by anti-P450d (P(3)450) were induced in both strains, but to a somewhat greater extent in the B6-Ahb mice. The hepatic accumulation of HCB at 15 weeks was greater in B6-Ahb than in B6-Ahd mice, in association with elevated hepatic lipid levels in the former strain. The results of this experiment indicate that the Ah locus influences the susceptibility of C57BL/6J mice to HCB-induced porphyria and are consistent with the suggestion that the sustained induction of P(3)450 and/or P(1)450 may be a causative factor in the development of this disease.

Animals↗

PubMed1988

Effects of SH-modifying reagents on the rat hepatic Ah receptor: inhibition of ligand binding and transformation, and disruption of the ligand-receptor complex.

The importance of sulfhydryl (SH) groups in maintenance of physicochemical properties of the rat hepatic Ah receptor was demonstrated using a variety of sulfhydryl (SH)-modifying reagents. Inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) specific binding was approximately equivalent by 5,5'-dithiobis(2-nitrobenzoate), mersalyl, N-ethylmaleimide, and p-chloromercuriphenylsulfonate, whose inhibition curves were steep in the concentration range close to that of nonprotein SH groups in cytosol (ED50 values 50-200 microM or 13-48 nmol/mg cytosolic protein). Inhibition by p-hydroxymercuribenzoate (PHMB), although exhibiting a lower ED50, was more gradual over this range; iodoacetamide was an order of magnitude less potent. The ability of dithiothreitol to reverse binding inhibition induced by 150 microM (approximately 60 nmol/mg protein) mersalyl diminished with time; it decreased more rapidly in the simultaneous presence of TCDD and mersalyl than when mersalyl was present alone, consistent with increased accessibility of key SH group(s) due to conformational changes attending TCDD-receptor complex formation. Brief exposure of unoccupied receptor to mersalyl prior to TCDD binding caused slower sedimentation of the complex in 0-KCl sucrose gradients and alterations in its elution profiles on DEAE- and DNA-Sepharose suggestive of some impairment of the transformation process. When reagents were added to the transformed TCDD-receptor complex, loss of binding was observed only at concentrations which were an order of magnitude higher than those inhibiting TCDD binding. Loss of binding by each reagent was biphasic, and except for that caused by mersalyl, was not complete even after 6-8 h. Dithiothreitol was able to reverse the effects of mersalyl or PHMB only partially and only if added during the early phase (10-30 min) of binding loss. Mersalyl was much more potent in disrupting the untransformed than the transformed TCDD receptor complex. Physical alteration of the mersalyl-treated TCDD-receptor complex was evident from gel filtration, sucrose gradients, and DNA- and DEAE-Sepharose chromatography. Our results are in striking contrast to the effects of these reagents on steroid receptors, whose bound steroid hormone ligand is rapidly and reversibly displaced by lower concentrations of reagent.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Influence of proteinase inhibitors and substrates on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding capacity of the rat hepatic Ah receptor.

These studies investigated the effects of various serine proteinase inhibitors and substrates on the TCDD-binding capacity of the rat hepatic Ah receptor. TCDD binding to the Ah receptor was inhibited by serine proteinase inhibitors phenylmethylsulfonyl fluoride (PMSF), tosyl-lysine chloromethyl ketone (TosLysCH2Cl), tosylamide-phenylethyl chloromethyl ketone (TosPheCH2Cl) and substrates tosyl-L-arginine methyl ester (TosArgOMe) and D-tryptophan methyl ester (TrpOMe). The order of potency was TosPheCH2Cl greater than TosLysCH2Cl much greater than PMSF approximately equal to TosArgOMe approximately equal to TrpOMe. Reactivity of the chloromethyl ketones with sulfhydryl groups was suggested by their steep inhibition curves above the concentration of nonprotein sulfhydryl groups, and the partial mitigation of inhibition by 1 mM dithiothreitol. Inhibition by these reagents was irreversible, while that by TosArgOMe and TrpOMe was completely reversible by gel filtration. The mechanism of inhibition by TosArgOMe and TrpOMe was formally competitive, with inhibition constants similar to those reported in steroid hormone receptor systems. Neither inhibitors nor substrates displaced previously bound TCDD.

Animals↗

PubMed1987

Changes in thyroid hormones and thyroxine glucuronidation in hamsters compared with rats following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, serum thyroxine (T4) is depressed. Since hamsters are relatively insensitive to TCDD-induced lethality, the effects of TCDD on several parameters of thyroid status were measured in hamsters as a comparison with the more sensitive rat. At 7 days after ip injection of TCDD, there was a dose-dependent increase in serum 3,5,3'-triiodothyronine (T3) and T4 in hamsters to a maximum level 200% of control; the ED50 was approximately 10 micrograms/kg. Hamsters receiving 100 micrograms/kg lost up to 4% of their body weight but began to recover after about 3 weeks. Serum T4 in these animals was elevated compared to pair-fed and ad libitum controls throughout the 53-day experiment, although it also began to recover after Day 21. This was in direct contrast to the marked reduction of T4 in rats exposed to lower doses of TCDD. T3 was significantly higher in TCDD-treated hamsters than in pair-fed controls on Days 2-7, and TSH was also elevated on Days 2-21. Reverse T3, like T4, was increased by TCDD in hamsters whereas it was decreased in rats. Hepatic microsomal UDP-glucuronosyltransferase (GT) activity was measured using T4 as substrate (T4-GT). On a whole liver basis, T4-GT was induced by TCDD by the same proportion in both rats and hamsters (170-180% of controls) although absolute activities in rats were 3- to 4-fold higher than in hamsters. This similarity in T4-GT inducibility by TCDD suggests that there are likely mechanisms in addition to T4-GT induction which account for the species-specific alterations in T4. Thus, while the response of thyroid hormones to TCDD differed qualitatively, effective doses in hamsters were higher than in rats, suggesting that these changes, although secondary, may correlate more directly with toxicity than does enzyme induction (whose ED50s are similar in both species). An understanding of the mechanism of this species difference may be helpful in unravelling the primary mechanisms of TCDD toxicity.

Animals↗

PubMed1987

Heterogeneity of the rat hepatic Ah receptor and evidence for transformation in vitro and in vivo.

The characteristics of the Ah receptor from rat liver were investigated following the incubation of cytosol with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under various conditions, and using DEAE- and DNA-Sepharose chromatography and sucrose density gradient centrifugation. These studies indicated that the Ah receptor can exist in three distinct forms in vitro that are dependent on the presence or absence of TCDD and the duration and temperature of incubation. The unoccupied receptor was distinguished by its elution from DEAE-Sepharose columns at 0.20-0.23 M NaCl and lack of affinity for DNA-Sepharose. Following the incubation of the unoccupied receptor with [3H]TCDD, two occupied forms were distinguished based on their overall surface charges and affinities for DNA. One of these forms was predominant following short incubations (2 h) with [3H]TCDD at a low temperature (0 degree C) and was characterized by having the same elution profile on DEAE-Sepharose as the unoccupied form, but demonstrated some affinity for DNA. Another occupied form was predominant following an incubation for a longer time (20 h, 0 degree C) or at an elevated temperature (2 h, 20 degrees C). This form had an overall surface charge that was less negative and a greater affinity for DNA. These changes in receptor characteristics were dependent on the presence of TCDD and were not accompanied by apparent changes in the sedimentation coefficients of the two occupied forms. Anion exchange chromatography of the [3H]TCDD-receptor complex extracted from hepatic nuclei of [3H]TCDD-treated rats indicated that the ligand-induced change of the unoccupied receptor to a less negatively charged form had occurred in vivo. These results indicated a biochemical heterogeneity of the Ah receptor and suggested the occurrence of a ligand- and temperature-dependent transformation process in vivo and in vitro.

Animals↗

PubMed1987

Characterization of the in vitro stability of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The in vitro stability of the Ah receptor from rat hepatic cytosol was evaluated by [3H]TCDD binding studies, gel filtration, and sucrose density gradient ultracentrifugation. Thermal inactivation of unoccupied receptor followed first-order kinetics between 5 and 40 degrees C, with an estimated Ea for inactivation of approximately 35 kcal/mol. Protease inhibitors did not reduce and dilution slightly increased the inactivation rate at 20 degrees C. Recovery and 20 degrees C stability decreased with increasing ionic strength. The TCDD-receptor complex was less susceptible to degradation at 20 degrees C, even in the presence of 0.4 M KCl. Specific binding was markedly pH dependent, with maximum recovery at 7.6. Analysis of the pH curve suggested that cysteine sulfhydryl groups may be involved in TCDD binding. Dithiothreitol (1 mM) maximized recovery and 20 degrees C stability, and addition of the thiol largely reactivated binding sites lost from cytosol prepared without it. Removal of low molecular weight components of cytosol by gel filtration resulted in a rapid 20 degrees C inactivation rate that could not be lessened by dithiothreitol. Glycerol (10% v/v) and EDTA (1.5 mM) maximized recovery of specific binding, but both decreased 20 degrees C stability in a concentration-dependent manner. Calcium chloride (4 mM) increased stability at 20 degrees C by approximately 20%, and retarded the characteristic shift in sedimentation coefficient from approximately 9 to approximately 6 S in high-salt sucrose gradients. The fact that sodium molybdate (20 mM) decreased recovery and 20 degrees C stability when dithiothreitol was present but slightly increased stability in its absence suggested an antagonism between the two compounds. Molybdate mitigated the inactivation induced by 0.4 M KCl, an effect which may be related to the observation of dual peaks in molybdate-containing high-salt sucrose gradients. These data indicate that thermal inactivation of the unoccupied rat hepatic Ah receptor primarily may be due to physical rather than enzymatic processes; (ii) sulfhydryl oxidation, removal of low molecular weight cytosolic components, and high ionic strength result in rapid rates of inactivation at 20 degrees C; and (iii) the large degree of protection conferred by TCDD binding implies a very tight ligand-receptor interaction, and as such accords with TCDDs extraordinary potency and persistence in producing its toxic and biochemical effects.

Animals↗

PubMed1987

alpha-Naphthoflavone antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced murine lymphocyte ethoxyresorufin-O-deethylase activity and immunosuppression.

Suppression of murine humoral immunity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to occur in vivo and in vitro. Studies have indicated that suppression of humoral immunity is mediated by the Ah receptor. Data presented in this paper demonstrate that alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF), like TCDD, bind to rat and murine hepatic and murine splenocyte cytosolic Ah receptor. Furthermore, BNF induces cytochrome P1-450 monooxygenase activity as measured by ethoxyresorufin-O-deethylase (EROD) in murine spleen cells to the same extent as TCDD. In contrast, ANF predominantly acts to antagonize TCDD induction of splenocyte EROD activity. Examination of humoral immunity in vitro demonstrated that BNF, like TCDD, is suppressive. Whereas ANF is suppressive at cytotoxic concentrations, lower concentrations of ANF antagonize the suppressive effect of TCDD. Antagonism by ANF of TCDD-induced EROD activity and suppression of humoral immunity occur at similar concentrations. These data suggest that ANF blocks TCDD suppression of B lymphocyte differentiation by competing with TCDD for binding to the Ah receptor. Since the mechanism of TCDD toxicity is not fully understood, probes such as ANF may be of great use in examining the role of the Ah receptor in mediating toxicity.

Animals↗

PubMed1986

Changes in hamster hepatic cytochrome P-450, ethoxycoumarin O-deethylase, and reduced NAD(P): menadione oxidoreductase following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Partial dissociation of temporal and dose-response relationships from elicited toxicity.

The temporal and dose-related characteristics of hepatic enzymes induced in the hamster by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined. Male Syrian golden hamsters received a single intraperitoneal injection of TCDD at a dose of 0-500 micrograms/kg. At various times up to 35 days, a number of variables were determined and compared: whole body, liver, and thymus weights; hepatic concentrations of cytochrome P-450 (P-450); and activities of 7-ethoxycoumarin O-deethylase (ECOD) and reduced NAD(P): menadione oxidoreductase (NMOR). Increased liver weights and decreased thymus weights were observed to be dose related. At day 7 following treatment, the approximate ED50 values for these responses were 15 and 100 micrograms/kg respectively. The ED50 values for the increase in hepatic P-450 concentrations and activities of ECOD and NMOR ranged from 0.5 to 2.0 micrograms/kg. At 10 and 500 micrograms/kg, NMOR activity remained maximally induced for up to 35 days. This was also the case for P-450 and ECOD activity at a dose of 10 micrograms/kg. At 500 micrograms/kg, both P-450 and ECOD demonstrated an induction up to day 4 followed by a decrease to near control levels by day 14. This decrease appeared to correlate with changes in hepatic morphology. These results demonstrate a dissociation of the induction of these hepatic enzymes from TCDD-induced lethality, in this species.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1986

Induction of cytochrome P-450 isozymes by hexachlorobenzene in rats and aromatic hydrocarbon (Ah)-responsive mice.

Hexachlorobenzene (HCB) differs markedly from other chlorinated benzenes (CBs) as an inducer of cytochrome P-450 (P-450) isozymes as determined by radioimmunoassay and immunoblotting. At greater than 99% pure, HCB induced both the phenobarbital-inducible forms, cytochromes P-450b + e (70 chi), and the 3-methylcholanthrene-inducible forms, cytochromes P-450c (58 chi) and P-450d (8 chi), in rat liver microsomes. The concentration of P-450d was considerably greater than that of P-450c in HCB-induced rat liver. In contrast to HCB, all lower chlorinated benzenes tested were PB-type inducers. Hexachlorobenzene increased the amounts of translatable messenger RNAs (mRNAs) for P-450b, P-450c, and P-450d in rat liver polysomes, suggesting that it increases the synthesis of these proteins. Evidence that HCB interacted with the putative Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was equivocal. Western blots of liver microsomes from Ah-responsive C57BL/6J (B6) and nonresponsive DBA/2J (D2) mice demonstrated that HCB produced a large increase in P3-450 and a very small increase in P1-450 in the responsive strain. The increase in P1-450 was not observed after HCB administration to nonresponsive mice, but a small increase in P3-450 was noted. These findings suggested that HCB may act through the Ah receptor. However, HCB was at best a very weak competitor for specific binding of [3H]-TCDD to the putative receptor in rat or mouse hepatic cytosol in vitro, producing decreases in binding of [3H]-TCDD only at very high concentrations (10(-6) to 10(-5) M).

Animals↗

PubMed1986

Toxicity and evidence for metabolic alterations in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated guinea pigs fed by total parenteral nutrition.

The effect of total parenteral nutrition (TPN) on the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male, Hartley-strain guinea pigs was determined. At a single dose of 2 micrograms TCDD/kg, TPN-fed guinea pigs maintained body weight at a level which was slightly, but consistently, below that of the TPN-fed control animals. However, despite the sustenance of body weight, TCDD-treated animals died or were sacrificed due to morbidity between Days 8 and 24 following treatment. Approximately 50% of this group demonstrated a profound loss of body weight within a few days prior to death or sacrifice. With the exception of the pattern of weight loss, the signs of toxicity in the TPN-fed, TCDD-treated animals were strikingly similar to those observed in TCDD-treated guinea pigs fed ad libitum. Although livers from TCDD-treated, TPN-fed animals demonstrated increased content of lipid and cytochrome P-450, this tissue appeared to be morphologically and functionally comparable to that from TPN-fed controls. Of the blood chemistry examined, only the serum concentrations of 3,5,3'-triiodothyronine were significantly decreased in the treated animals fed by TPN. Results were also compared to TCDD-treated guinea pigs fed ad libitum and respective pair-fed controls. Many of the physiological and biochemical responses observed in animals fed ad libitum following TCDD treatment could be explained by a decrease in food consumption. This study demonstrated that although food consumption clearly accounts for the major effect of TCDD on body weight loss in guinea pigs fed ad libitum, additional physiological and/or biochemical alterations occurred which also contribute to body weight loss, other signs of toxicity, and subsequent lethality.

Animals↗

PubMed1986

Growth and hepatic composition in the guinea pig after long-term parenteral hyperalimentation.

This study examined the feasibility of maintaining male Hartley guinea pigs on long-term hyperalimentation. Data from animals fed ad libitum, but infused with 0.9% saline, indicated that there was minimal effect from catheter implantation, sepsis, or the infusion of large volumes of fluid. This group compared with animals fed and watered ad libitum demonstrated a nearly identical growth rate (6.33 +/- 1.18 and 6.12 +/- 1.32 g/day, respectively), serum chemistry, tissue weights, and hepatic composition and morphology. Animals infused with a total parenteral diet demonstrated growth rates of 4.06 +/- 1.46 g/day for up to 25 days. Loss of infused animals was due in varying degrees to sepsis, mechanical failure, improper placement of the cannula, loss of patency, and death from unknown causes. Morphological analysis of animals fed by total parenteral nutrition revealed an altered distribution and increased size of lipid droplets in hepatic parenchymal and Kupffer cells and glycogen accumulation by the parenchymal cells. Decreased hepatic content of total protein and lipid, as well as cytochrome P450, was also observed. Similarly, serum values of triglyceride were decreased in animals fed by the total parenteral diet. This study indicated that the guinea pig fed by hyperalimentation may be a useful animal model for a number of clinical and basic research applications.

Animals↗

PubMed1986

Structure-activity relationships of chlorinated benzenes as inducers of hepatic cytochrome P-450 isozymes in the rat.

This study compared the ability of hexachlorobenzene (HCB) and of other chlorinated benzenes to induce cytochrome P-450 isozymes in rat liver. HCB (greater than 99% pure) induced both the phenobarbital-inducible forms (cytochrome P-450b and P-450e) and the 3-methylcholanthrene (3-MC)-inducible forms (P-450c and P-450d) of cytochrome P-450. However, HCB differed from many 3-MC-type inducers by inducing P-450d preferentially over P-450c. In contrast to HCB, the lower chlorinated benzenes did not induce significant amounts of P-450c or P-450d in the rat, but were phenobarbital-type inducers, inducing P-450b and P-450e. These data indicate that the hepatic effects of HCB differ markedly from those of other chlorinated benzenes. However, chlorinated dibenzodioxins also induce P-450c and P-450d in the rat, and although chlorinated dibenzodioxins and dibenzofurans contaminate certain commercial products, none were detected by gas chromatography/mass spectrometry (detection limit 0.5 ppm) in the HCB used in this study. The evidence that HCB interacted with the receptor for 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) was equivocal. At a concentration of 10(-6) M, HCB produced a slight decrease (18%) in the binding of 3H-TCDD to this protein in vitro, but had no effect at lower concentrations. However, as an inducer of two 3-MC-inducible isozymes of P-450, HCB was clearly more effective in aromatic-hydrocarbon-responsive mice (C57Bl/6J) than in non-responsive mice (DBA/2J), suggesting that HCB may act through the Ah receptor.

Animals↗

PubMed1984

Examination and rapid analysis of hepatic cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using gel-permeation high performance liquid chromatography.

Gel-permeation high performance liquid chromatography was used to examine the molecular characteristics of the cytosolic and nuclear receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin. The physical properties for this receptor previously established by sucrose-density gradient analysis have been maintained by the technique. The high performance liquid chromatography method offers the advantages of rapid separation and reproducibility thus minimizing possible effects of proteolytic enzymes or dissociation of the ligand-receptor complex. The specific binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin as measured by this system also corresponds quantitatively to that determined by the hydroxyapatite method. These methods will facilitate a further understanding of the biochemical actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, as well as an understanding of the physiological function of this receptor molecule.

Animals↗

PubMed1984

Ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver, lung, and thymus.

The ontogeny of the cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin was studied in Sprague-Dawley rats by quantitation of the receptor in hepatic, lung, and thymic cytosol. Concentrations of hepatic and lung cytosolic receptors increased rapidly after birth and remained at the highest levels from days 2 to 21. After this time, receptor levels in these tissues slowly declined with age. In the thymus, cytosolic receptor concentrations remained high from days 2 to 42 following birth. In these tissues and at all times examined, the receptors demonstrated very high affinities for [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. From days 15 to 42 following birth, no consistent sex related differences in receptor content or affinity were observed in any of these tissues.

Aging↗

PubMed1984

Cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species.

The presence and properties of the Ah receptor were examined in the guinea pig, rat, hamster, monkey, and three different strains of mice. These species and strains have demonstrated differences in sensitivity and variability of response to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. All species examined, with the exception of DBA/2J mice, possess similar amounts of binding protein with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin in hepatic tissue. Numerous dibenzo-p-dioxin congeners and polycyclic aromatic hydrocarbons demonstrated a similar rank order ability to bind to receptor molecules from these species. When analyzed by gel-exclusion high-performance liquid chromatography, hepatic cytosolic receptors from all species eluted at volumes corresponding to a similar molecular weight range. Association of the hepatic Ah receptor with the nuclear fraction was observed in all cases following the i.p. treatment of guinea pig, rat, C57BL/6J mouse, or hamster with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. In all species and tissues examined, with the exception of hamster duodenum and thymus, the highest concentrations of receptor were localized in the liver, lung, thymus, intestine, and kidney. Exceptionally high concentrations of receptors were also observed in guinea pig testes. These findings indicate that, despite species and tissue specific differences in the biochemical and toxicological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, a number of different mammalian species possess Ah receptors with similar properties. Thus, the correlative differences between certain strains of mice in terms of altered specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and sensitivity to this compound may be unique and not necessarily applicable to other species. Although all data indicate that the receptor mediates these responses, it appears that species- and tissue-specific differences may be determined by a number of additional factors. These results also suggest the conservation of some, as yet unknown, functional role of the receptor molecule.

Animals↗