Enhancement of toxicity and enzyme-repressing activity ofp-dioxane by chlorination: Stereoselective effects

The acute toxicity of p-dioxane may be enhanced up to 1000-fold by chlorination of the compound. The effect was stereoselective. Of the stereoisomers tested, tetrachloro-p-dioxane, isomer I (2r, 3t, 5t, 6c) was over 80 times more toxic than isomer II (2r, 3c, 5t, 6t). The latter compound was also a potent repressor of hepatic dimethylnitrosamine-demethylase I (DMN-d) and aryl hydrocarbon hydroxylase (AHH).     

The Ah regulatory gene product. Survey of nineteen polycyclic aromatic compounds' and fifteen benzo[a]pyrene metabolites' capacity to bind to the cytosolic receptor

The capacity of 19 polycyclic aromatic compounds and 15 benzo[a]pyrene metabolites to displace $\text{[1,6-}{}^3\text{H}\text{]2,3,7,8-}\text{tetrachlorodibenzo}\text{-p-}\text{dioxine}\text{([}{}^3\text{H]Tcdd}\text{)}$ from the mouse liver cytosolic Ah receptor was examined. We compared our data with various parameters taken from previously published results: the capacity of seven polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase (AHH) activity in human cell cultures, the capacity of 10 polycyclic hydrocarbons to induce azo dye N-demethylase activity in rat liver, the capacity of 6 polycyclic hydrocarbons to shorten zoxazolamine paralysis times in the intact rat, and the capacity of 15 benzo[a]pyrene metabolites to induce AHH activity in rat hepatoma H-4-II-E cultures. An excellent correlation is seen between the capacity to displace the radioligand from the Ah receptor and the capacity to induce these monooxygenase activities. Differences in the rate of cellular uptake and formation of alkali-extractable metabolites of dibenzo[a,h]anthracene, 3-methylcholanthrene, and benzo[a]anthracene in Hepa-1 mouse hepatoma cell cultures do not account for differences in the capacity of these three polycyclic hydrocarbons to displace [³H]TCDD from the Ah receptor.     

Effects of pentachlorophenol on rat liver changes induced by hexachlorobenzene,with special reference to porphyria,and alterations in mixed function oxygen ases

Hexachlorobenzene (HCB, 1000 ppm) and 500 ppm pentachlorophenol (PCP) were fed separately or in combination to female Wistar rats. A control group was provided with standard food without HCB or PCP. Subgroups of 4 rats were killed after 1,2,4,6 and 8 weeks. No significant difference was found between the amounts of HCB accumulated in the livers of the HCB and HCB + PCP fed rats. Administering HCB together with PCP caused a noticeable accumulation of PCP in the liver, compared to the results after administering HCB and PCP separately. In the HCB and HCB + PCP fed groups liver weight increased continuously during the experiments. Microsomal cytochrome P-450, NADPH-cytochrome c reductase, ethoxyresorufin O-de-ethylase, aminopyrine N-demethylase, and glucuronyl transferase increased to a maximum in 2–4 weeks in HCB and HCB + PCP fed rats. Pentachlorophenol accelerates the onset of HCB porphyria, in other words it increases the total urinary porphyrin excretion and causes an earlier disturbance of the porphyrin pattern.     

Genetically mediated induction of aryl hydrocarbon hydroxylase activity in mice by polychlorinated dibenzofuran isomers and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Hepatic aryl hydrocarbon hydroxylase (AHH)-inducing potency of toxic polychlorinated aromatic hydrocarbons such as polychlorinated dibenzofurans (PCDFs), 3,4,5,3,4,5-hexachlorobiphenyl (HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in four inbred strains of mice with different phenotypes of Ah locus, i.e., AHH-responsive strains: C57BL/6N and AKR/Ms Qdj, and AHH-nonresponsive strains: DBA/2Cr Slc and Qdj; DDD. Eight individual PCDF isomers or TCDD were administered IP in doses of 30 g/kg; HCB was given in a dose of 120 g/kg. In AHH-nonresponsive strains of mice, only TCDD significantly induced hepatic AHH activity, while in AHH-responsive strains, 2,3,7,8-tetrachlorodibenzofuran(2,3,7,8-TCDF), 1,2,3,7,8-pentachlorodibenzofuran(1,2,3,7,8-PCDF) 2, 3, 4, 7, 8-pentachlorodibenzofuran (2, 3,4, 7, 8-PCDF), and TCDD significantly enhanced the enzyme activity, and the induced AHH activities with the three PCDF isomers were about 30–65% of those of TCDD. These results indicate that AHH responsiveness in mice segregates with the induction of AHH activity by PCDF isomers and may also segregate with the toxic potency of the isomers; i.e., toxic potencies of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF in AHH-responsive strains of mice may be much greater than those in AHH-nonresponsive strains of mice. Taking into account both the potent AHH inducibility and the high bioaccumulation of 2,3,7,8-TCDF, 1,2,3,7,8-PCDF, and 2,3,4,7,8-PCDF, these three PCDF isomers should be given greater attention with regard to environmental contamination.A part of this work was presented at the 41st annual meeting of the Japanese Society of Public Health, October 27–29, 1982, Fukuoka, Japan.     

Genetic differences between C57BL/6 and DBA/2 mice in the inductions of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene,p-nitrophenol,and bilirubin by 3-methylcholanthrene

In C57BL/6 mice, aryl hydrocarbon hydroxylase (AHH) increased 1 day after treatment with 3-methylcholanthrene, and induction of UDP-glucuronyl transferases for 3-hydroxybenzo(a)pyrene, p-nitrophenol and bilirubin in the liver microsomes was observed 2 to 5 days later. In DBA/2 mice, neither AHH nor transferase activities were influenced by 3-methylcholanthrene. These results suggest that induction of activating and detoxicating enzymes is genetically linked.     

Time course of the induction of aryl hydrocarbon hydroxylase in rat liver nuclei and microsomes by phenobarbital, 3-methylcholanthrene, 2,3,7,8-tetrachloro-dibenzo-p-dioxin,dieldrin and other inducers

Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat liver nuclei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, 0.01 mg/kg) with an induction to 500 and 300 per cent for nuclei and microsomes, respectively, after 2 days, and to 400 per cent for both after 12 days. Phenobarbital (PB) was given continuously in the drinking water (1 g/l) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. the nuclear activity was only slightly induced to a Constant level of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daily i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down to control values after 12 days. Other inducers tested were benz[a]anthracene (BA), hexachlorobenzene (HCB) and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The induction pattern with BA was similar to that of MC, a model compound for the group of cytochrome P448 inducers. the induction by HCB and DDT resembled that by PB, a typical cytochrome P450 inducer.     

Pleiotropic effect of the gene hairless on hepatotoxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin in mice

Treatment of mice of the A2G-hr/+ congenic line with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in the development of hepatic porphyria over a period of 4 weeks. Female mice responded to a lesser extent than did males. The degree of porphyria in haired heterozygotes (hr/+) was less than in the corresponding hairless homozygotes (hr/hr) and the haired mice had lower resting metabolic rates than hairless mice. Adaptation of mice of either genotype to a 32–33 °C environment resulted in a decrease in resting metabolic rate and a reduction in hepatic porphyrin levels. Histologically-demonstrated necrotic changes in livers were accompanied by increased activity of alanine aminotransferase and sorbitol dehydrogenase in the plasma; however, there was no clear temporal trend in plasma enzyme levels. Elevated environmental temperature reduced the plasma alanine aminotransferase activity. The study provided evidence for a pleiotropic effect of variation at the hr locus being expressed in TCDD hepatotoxicity. Suggestions for mechanisms whereby the effect can be mediated through alterations in resting metabolic rate are made.     

Induction of Cyp1a1 and Cyp1b1 and formation of DNA adducts in C57BL/6, Balb/c, and F1 mice following in utero exposure to 3-methylcholanthrene

Fetal mice are more sensitive to chemical carcinogens than are adults. Previous studies from our laboratory demonstrated differences in the mutational spectrum induced in the Ki-ras gene from lung tumors isolated from [D2 x B6D2F1]F2 mice and Balb/c mice treated in utero with 3-methylcholanthrene (MC). We thus determined if differences in metabolism, adduct formation, or adduct repair influence strain-specific responses to transplacental MC exposure in C57BL/6 (B6), Balb/c (BC), and reciprocal F1 crosses between these two strains of mice. The induction of Cyp1a1 and Cyp1b1 in fetal lung and liver tissue was determined by quantitative fluorescent real-time PCR. MC treatment caused maximal induction of Cyp1a1 and Cyp1b1 RNA 2-8 h after injection in both organs. RNA levels for both genes then declined in both fetal organs, but a small biphasic, secondary increase in Cyp1a1 was observed specifically in the fetal lung 24-48 h after MC exposure in all four strains. Cyp1a1 induction by MC at 4 h was 2-5 times greater in fetal liver (7000- to 16,000-fold) than fetal lung (2000- to 6000-fold). Cyp1b1 induction in both fetal lung and liver was similar and much lower than that observed for Cyp1a1, with induction ratios of 8- to 18-fold in fetal lung and 10- to 20-fold in fetal liver. The overall kinetics and patterns of induction were thus very similar across the four strains of mice. The only significant strain-specific effect appeared to be the relatively poor induction of Cyp1b1 in the parental strain of B6 mice, especially in fetal lung tissue. We also measured the levels of MC adducts and their disappearance from lung tissue by the P(32) post-labeling assay on gestation days 18 and 19 and postnatal days 1, 4, 11, and 18. Few differences were seen between the different strains of mice; the parental strain of B6 mice had nominally higher levels of DNA adducts 2 (gestation day 19) and 4 (postnatal day 1) days after injection, although this was not statistically significant. These results indicate that differences in Phase I metabolism of MC and formation of MC-DNA adducts are unlikely to account for the marked differences observed in the Ki-ras mutational spectrum seen in previous studies. Further, the results suggest that other genetic factors may interact with chemical carcinogens in determining individual susceptibility to these agents during development.     

Genetically mediated induction of aryl hydrocarbon hydroxylase activity in human lymphoblastoid cells by polychlorinated dibenzofuran isomers and 2,3,7,8-tetrachlorodibenzo-p-dioxin

Aryl hydrocarbon hydroxylase(AHH)-inducing potency of toxic polychlorinated aromatic hydrocarbons such as polychlorinated dibenzofuran (PCDF) isomers, 3,4,5,3,4,5-hexachlorobiphenyl (HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated in human lymphoblastoid cell lines with different AHH inducibility for 3-methylcholanthrene (3-MC) obtained from healthy subjects. Each of the cell lines was treated with eitht individual PCDF isomers, TCDD, and HCB at doses of 1.9–15 ng/ml of culture medium, 1.9–7.5 ng/ml and 95 ng/ml, respectively. Lymphoblastoid cell lines were arbitrarily classified into three groups based on their AHH inducibilities with 3-MC (2.5 M); low (3-MC/ control=I<3), middle (3<=I<6) and high (I>=6). Degrees of the enzyme inducibilities of the organochlorine compounds proportionally increased with those for 3-MC. AHH inducibilities with 2,3,4,7,8-pentachlorodibenzofuran(2,3,4,7,8-PCDF), 1,2,3,4,6,7-hexachlorodibenzofuran(1,2,3.4,6,7-HCDF) and 1,2,3,4,7,8-hexachlorodibenzofuran(1,2,3,4,7,8-HCDF) were comparable to those of TCDD at doses of 7.5 ng/ ml, and about twice as high as those of 2,3,7,8-tetrachlorodibenzofuran (TCDF), at the same dose, HCB, at a dose of 95 ng/ ml, did not induce enzyme activity. The experimental evidence indicated that AHH inducibility by the organochlorine compounds reflected the genetic susceptibility of the cells to the phenomenon of induction, and PCDF isomers found at relatively high concentrations in tissues of mammals exerted the highest values of AHH induction.Part of this work was presented at the 53rd annual meeting of the Japanese Society for Hygiene, April 5–7, 1983, Osaka, Japan     

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K_m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V_max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V_max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V_max using BOX was enhanced after TCDD administration only. The K_m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K_m using STOX, while it increased the K_m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.     

Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2

Adult female Fisher 344 rats received drinking water containing 0, 4, 40, 100, or 200 parts per million of dimethylarsinic acid or 100 parts per million of arsenate for 14 days. Urine was collected during the last 24 h of exposure. Tissues were then taken for analysis of dimethylated and trimethylated arsenicals; urines were analyzed for these arsenicals and their thiolated derivatives. In dimethylarsinic acid-treated rats, highest concentrations of dimethylated arsenic were found in blood. In lung, liver, and kidney, concentrations of dimethylated arsenic exceeded those of trimethylated species; in urinary bladder and urine, trimethylated arsenic predominated. Dimethylthioarsinic acid and trimethylarsine sulfide were present in urine of dimethylarsinic acid-treated rats. Concentrations of dimethylated arsenicals were similar in most tissues of dimethylarsinic acid- and arsenate-treated rats, including urinary bladder which is the target for dimethylarsinic acid-induced carcinogenesis in the rat. Mean concentration of dimethylated arsenic was significantly higher (P<0.05) in urine of dimethylarsinic acid-treated rats than in arsenate-treated rats, suggesting a difference between treatment groups in the flux of dimethylated arsenic through urinary bladder. Concentrations of trimethylated arsenic concentrations were consistently higher in dimethylarsinic acid-treated rats than in arsenate-treated rats; these differences were significant (P<0.05) in liver, urinary bladder, and urine. Concentrations of dimethylthioarsinic acid and trimethylarsine sulfide were higher in urine from dimethylarsinic acid-treated rats than from arsenate-treated rats. Dimethylarsinic acid is extensively metabolized in the rat, yielding significant concentrations of trimethylated species and of thiolated derivatives. One or more of these metabolites could be the species causing alterations of cellular function that lead to tumors in the urinary bladder.     

Effects of aryl hydrocarbon receptor null mutation and in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on prostate and seminal vesicle development in C57BL/6 mice.

Experiments were conducted to determine the effects of aryl hydrocarbon receptor (AhR) null mutation and in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, alone and in combination, on prostate and seminal vesicle development in C57BL/6 mice. AhR heterozygous (Ahr+/-) mice were mated, and pregnant females were dosed orally on gestation day 13 with TCDD (5 microg/kg) or vehicle. Pups underwent necropsy on postnatal days (PNDs) 35 and 90. Comparison of vehicle-exposed AhR knockout (AhRKO;Ahr-/-) with wild-type (Ahr+/+) pups revealed that the AhR is necessary for normal dorsolateral prostate, anterior prostate, and seminal vesicle development but apparently not for ventral prostate development. In wild-type mice,in utero and lactational TCDD exposure reduced ventral prostate weight by 79-87% and mRNA expression for its major androgen-dependent secretory protein (MP25) by 99%. Yet high levels of mRNA for a secretory protein normally produced primarily by the lateral prostate (PSP94) were expressed. These effects were predominantly AhR dependent because TCDD had little if any effect in AhRKO mice. TCDD reduced dorsolateral prostate weight in wild-type but not AhRKO mice and had no significant effect on expression of mRNA for PSP94 or for probasin, a major androgen-dependent secretory protein. The PSP94 results suggest that TCDD may have caused a respecification of prostatic gene expression. TCDD reduced anterior prostate weight by more than half, and expression of mRNA for its major androgen-dependent secretory protein (renin-1) was greatly reduced. These effects were AhR dependent. Seminal vesicle weight was reduced by TCDD in wild-type mice but was increased in AhRKO mice on PND 35 and decreased on PND 90 (relative weight only). Androgen receptor mRNA levels were not significantly altered in any prostate lobe, and all organs appeared histologically normal in all groups. Serum testosterone concentrations were unchanged, and modest reductions in serum 5alpha-androstane-3alpha,17beta-diol concentrations could not account for the effects on sex organs. Collectively, these results indicate that the AhR signaling pathway plays a role in normal accessory sex organ development and thatin utero and lactational TCDD exposure disrupts development of these organs via spatially and perhaps temporally specific mechanisms.     

Protection by indole-3-carbinol against covalent binding of benzo[a]pyrene metabolites to mouse liver DNA and protein

Indole-3-carbinol (I-3-C) is a compound present in many cruciferous vegetables that has been shown to reduce aryl hydrocarbon-induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the activity of hepatic aryl hydrocarbon hydroxylase (AHH), and its ability to inhibit the covalent binding of benzo[a]pyrene (BaP) metabolites to DNA and protein. Using an in vitro system and a hepatic postmitochondrial fraction from mice that had been treated by gavage with I-3-C, we found that up to 90% of the covalent binding of BaP metabolites to macromolecules was eliminated, while AHH activity was unchanged. In experiments in vivo, treatment of mice by gavage with I-3-C before [¹⁴C]BaP resulted in up to an 80% decrease in covalent binding of ¹⁴C to DNA or protein with no concomitant decrease in hepatic AHH activity. These results suggest that I-3-C administered in vivo confers protection against the binding of BaP oxidation products to hepatic cellular macromolecules.     

Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Halogenated agonists for the aryl hydrocarbon receptor (AHR), such as 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), cause developmental toxicity in fish. AHR dependence of these effects is known for TCDD but only presumed for PCB126, and the AHR-regulated genes involved are known only in part. We defined the role of AHR in regulation of four cytochrome P450 1 (CYP1) genes and the effect of PCB126 on cell cycle genes (i.e., PCNA and cyclin E) in zebra fish (Danio rerio) embryos. Basal and PCB126-induced expression of CYP1A, CYP1B1, CYP1C1, and CYP1C2 was examined over time as well as in relation to cell cycle gene expression and morphological effects of PCB126 in developing zebra fish. The four CYP1 genes differed in the time for maximal basal and induced expression, i.e., CYP1B1 peaked within 2 days postfertilization (dpf), the CYP1Cs around hatching (3 dpf), and CYP1A after hatching (14-21 dpf). These results indicate developmental periods when the CYP1s may play physiological roles. PCB126 (0.3-100nM) caused concentration-dependent CYP1 gene induction (EC50: 1.4-2.7nM, Lowest observed effect concentration [LOEC]: 0.3-1nM) and pericardial edema (EC50: 4.4nM, LOEC: 3nM) in 3-dpf embryos. Blockage of AHR2 translation significantly inhibited these effects of PCB126 and TCDD. PCNA gene expression was reduced by PCB126 in a concentration-dependent manner, suggesting that PCB126 could suppress cell proliferation. Our results indicate that the four CYP1 genes examined are regulated by AHR2 and that the effect of PCB126 on morphology in zebra fish embryos is AHR2 dependent. Moreover, the developmental patterns of expression and induction suggest that CYP1 enzymes could function in normal development and in developmental toxicity of PCB126 in fish embryos.     

Characterization of the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in B6C3F1 and DBA/2 mice following single and repeated exposures

Previous studies have demonstrated that repeated (14 day) administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances the suppression of humoral immunity in DBA/2 (Ah-low responder) mice relative to the effect seen with identical cumulative doses after a single treatment (cumulative doses of 4.2, 14.0, and 42 mg/kg). In the present studies, we have explored this phenomenon further by determining the status of several specific parameters, which might account for the increase in antibody suppression in the DBA/2 strain following repeated TCDD exposures. Included in these studies was the induction of hepatic and splenic microsomal 7-ethoxyresorufin-o-deethylase (EROD; P4501A1) activity and biodistribution of the administered TCDD into various target organs and tissues. Changes in lymphocyte subpopulations within the spleen were also assessed by flow cytometry following both single and repeated dosing. All studies made use of direct comparisons between DBA/2 (Ah-low responder) and B6C3F1 (Ah-high responder) female mice. Results of these studies demonstrate that the enhanced suppression of humoral immunity in DBA/2 mice following repeated exposure to TCDD is not directly associated with increases in liver microsomal EROD activity and does not appear to be correlated with changes in the pattern of biodistribution or amount of TCDD within the liver or spleen of these animals. In contrast, the most significant changes that occurred following repeated dosing in either strain were observed in the levels of microsomal EROD activity and immune cell ratios within the spleen. This effect was characterized as an increase in microsomal EROD activity, and a corresponding reduction in the numbers of a non-B/non-T cell population in the spleen. Received: 5 June 1997 / Accepted: 26 August 1997     

Effect of dietary indole-3-carbinol on intestinal and hepatic monooxygenase, glutathione S-transferase and epoxide hydrolase activities in the rat

Male Sprague-Dawley rats were fed on purified diets supplemented with 50–500 ppm indole-3-carbinol (I3C), a compound present in cruciferous vegetables, or with 25% Brussels sprouts (Brassica oleracea) for 10 days after a 1-wk equilibration on a purified diet. Cytosolic and microsomal fractions were prepared from liver and intestinal mucosae. Intestinal aryl hydrocarbon hydroxylase (AHH) activity was increased significantly (P < 0.05) over basal levels by I3C at 50 ppm (a 6.1-fold increase), at 125 ppm (11.8-fold), at 250 ppm (14.1-fold) and at 500 ppm (20.2-fold) and by 25% Brussels sprouts (3.6-fold). Intestinal ethoxycoumarin O-deethylase (ECD) activity was also significantly increased by I3C, the increases being 4.6-, 8.7-, 9.3- and 11.2-fold with 50, 125, 250 and 500 ppm I3C, respectively, and 3.2-fold with the sprouts diet. Hepatic AHH and ECD were not increased significantly by any of these dietary treatments. Hepatic and intestinal glutathione S-transferase (GST) activities were increased (P < 0.05) 1.9- and 1.6-fold, respectively, by the sprouts diet but were not significantly affected even by 500 ppm I3C. Microsomal epoxide hydrolase (EH) activity of the small intestine was increased 2.0-fold by the sprouts diet but was unaffected by I3C. Hepatic cytochrome P-450 was increased 1.3-fold by the sprouts diet although I3C at 500 ppm only produced a 1.1-fold increase. A no-effect-level for I3C on intestinal monooxygenase induction was estimated to be between 16 and 25 ppm, supporting the contention that I3C can account for much of the monooxygenase induction observed when experimental animals are fed diets high in cruciferous vegetables. The results also indicate that Brassica oleracea contains other compounds which are responsible for the induction of GST and EH activities.     

Elevated binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene to the Ah receptor in hepatic cytosols from phenobarbital-treated rats and mice

Binding of 3-methylcholanthrene (MC),2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and other “MC-type” inducers to cytosolic Ah receptor sites is the first specific step in induction of aryl hydrocarbon hydroxylase (AHH; cytochrome P₁?450) by these compounds. [₃H]TCDD and [₃H]MC were used as radioligands to quantitate and characterize Ah receptor in hepatic cytosols from genetically “responsive” C57BL/6J mice, genetically “nonresponsive” DBA/2J mice, and AHH-inducible Sprague-Dawley rats. Injection of 50–100 mg/kg of phenobarbital (PB) for 3 days more than doubled the concentration of Ah receptor in hepatic cytosol from Sprague-Dawley rats. In C57BL/6J mice, PB injection at 25 mg/kg × 3 days significantly increased (P < 0.01) the Ah receptor concentration in hepatic cytosol. No cytosolic Ah receptor was detectable in hepatic cytosol from untreated DBA/2J mice, nor did any Ah receptor appear after PB treatment in this “nonresponsive” strain. Although PB significantly elevated Ah receptor in hepatic cytosols of responsive rodents, many previous studies have shown that the maximal level of AHH activity in animals given PB and an “MC-type” inducer simultaneously is additive rather than synergistic. Ah receptor concentrations can be doubled by PB treatment without doubling the subsequent AHH-induction response to “MC-type” compounds. Thus, the cytosolic Ah receptor concentration per se may not be the primary determinant of a given tissue's maximal capacity for AHH induction by “MC-type” compounds.     

Effect of maternal cigarette smoke exposure on foetuses of inbred strains of mice responsive and non-responsive to polycyclic aromatic hydrocarbons

To evaluate the role of genetically controlled responsiveness to polycyclic aromatic hydrocarbons (PAH) in the effect of cigarette smoke on birth weight, pregnant mice were exposed to cigarette smoke from day 1 to day 17 of pregnancy and the offspring studied on day 18. An unequivocal phenotyping of foetuses with respect to responsiveness was achieved by measuring the activity of aryl hydrocarbon hydroxylase (AHH) in the foetal liver and corresponding placenta after pretreatment of the dam with β-naphthoflavone. The foetuses of dams exposed to standard non-filter cigarettes were consistently smaller than those of the controls, whereas smoke from commercial filter cigarettes was only marginally effective. No statistically significant association with the responsiveness to PAH could be found with respect to this effect on weight. These results suggest that genetically determined responsiveness to PAH, although of importance for PAH foetotoxicity in mice, is not important in the effect of cigarette smoke on birth weight in the strains studied.     

The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2 (-/-) knockout and C57BL/6N parental strains of mice

Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ratios. This study was initiated to determine whether TCDD was inherently poorly metabolized or unavailable for metabolism because of sequestration to CYP1A2. [³H]TCDD was administered as a single, oral dose (0.1 and 10 μg/kg) to 12 male C57BL/6N mice or 12 CYP1A2 (-/-) mice. At 96 h, less than 5% of the dose was eliminated in the urine of all groups, and TCDD detected in urine was bound to mouse major urinary protein (mMUP). Feces were the major elimination pathway (24-31% of dose), and fecal extracts and non-extractables were quantitated by HPLC for metabolites. No great differences in urinary or fecal elimination (% dose) were observed between the high and low dose treatments. TCDD concentrations were the highest in adipose tissue for CYP1A2 knockout mice but in liver for C57BL/6N mice supporting the role of hepatic CYP1A2 in the sequestration of TCDD. Overall metabolism between parental and knockout strains showed no statistical differences at either the high or low doses. The data suggested that metabolism of TCDD is inherently slow, due principally to CYP1A1, and that hepatic CYP1A2 is not an active participant in the metabolism of TCDD in male mice. Rather, CYP1A2 governs the pharmacokinetics of TCDD by making it unavailable for hepatic CYP1A1 through sequestration and attenuating extrahepatic tissue disposition.     

Abnormal expression of MAPK, EGFR, CK17 and TGk in the skin lesions of chloracne patients exposed to dioxins

Objective

Chloracne is one of the most sensitive and specific hallmark of dioxin intoxication. Although its clinical features are clearly described, poor understanding of the molecular pathways of dioxin-induced chloracne hampers a rational approach to therapy. The aim of the present study was to investigate the role of EGFR, MAPK, CK17, and TGk in the pathogenesis of chloracne related to dioxin exposures.

Methods

Epidermal tissues of twelve chloracne patients exposed to dioxins were compared with tissues from 12 healthy controls. These skin tissues were obtained by punch biopsies. p-EGFR and p-MAPK were examined by immunofluorescence. The mRNA and protein levels of CK17 and TGk were examined by fluorescence in situ hybridization and immunohistochemistry, respectively.

Results

p-EGFR and p-MAPK were found in all chloracne tissues, whereas no expression was found in the controls. CK17 mRNA and protein were also found in all chloracne lesions, but none in controls (P = 0.000). TGk mRNA and protein were detected in both groups, but the distribution was distinct. The positive signals in the controls were mainly in the stratum granulosum, while in the chloracne tissues, the positive signals were found more significantly in the stratum granulosum and stratum spinosum.

Conclusions

The results demonstrate that in the human skin the activation of mitogen-activated protein kinase pathway and up-regulation of CK17 and TGK may play roles in the pathogenesis of chloracne related to dioxin exposures.