Jean E. Francis

Incomplete correlation of 2,3,7,8-tetrachlorodibenzo-p-dioxin hepatotoxicity with Ah phenotype in mice☆

Pretreatment of male mice of the inbred strains A2G, BALB/c, C57BL/10, and AKR with iron dextran synergized the action of a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 75 micrograms/kg) in causing hepatic porphyria and necrosis 35 days later. There was no effect in DBA/2 mice. Increased porphyrin levels were associated with decreased hepatic activity of uroporphyrinogen decarboxylase. Iron alone had no effect on porphyrin levels or decarboxylase activity. In male BALB/c mice given TCDD alone there was a delay in the onset of porphyria. Female BALB/c, AKR, and AKR X DBA/2 F1 mice were more resistant to the porphyrinogenic effect of TCDD than males. Development of porphyria did not correlate with Ah phenotype of the mice. The inheritance of sensitivity to TCDD in crosses of the AKR and DBA/2 strains, both Ah nonresponsive, was studied by a biometrical genetic analysis. The inheritances of increased porphyrin levels and of increased plasma activity of enzymes indicative of hepatic necrosis were both complex. Segregation of alleles at more than one locus was required to explain the data. A lack of correlation of porphyrins with plasma enzyme levels in the F2 generation suggested that the expression of these traits was determined independently. Genes other than Ah influence the development of TCDD-induced hepatotoxicity in mice.

Oxidation of uroporphyrinogens by hydroxyl radicals Evidence for nonporphyrin products as potential inhibitors of uroporphyrinogen decar☐ylase

A hydroxyl radical‐generating system, hypoxanthine/xanthine oxidase/Fe‐EDTA, oxidises uroporphyrinogens to uroporphyrins and to more polar nonporphyrin products. The evidence suggests that the nonporphyrin products have inhibitory activity towards mouse liver uroporphyrinogen decar☐ylase which could explain some forms of human and experimental porphyrias.

Hepatic toxicity and uroporphyrinogen decarboxylase activity following a single dose of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin to mice

Abstract A single, low-lethality, oral dose of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) (75 μg/kg) induces an hepatic porphyria in C57BL/10 mice of either sex. The hepatic porphyrin levels are maximal 4–6 weeks after dosing and are still elevated 12 weeks after the dose. The activity of hepatic uroporphyrinogen decarboxylase is depressed within one week of the dose and this appears to precede the onset of porphyria. Mice of the DBA/2 strain show no changes of the same magnitude at doses of 1200 μg/kg, for porphyrin levels, or 75 μg/kg, for decarboxylase activity. In both strains there is an increase in hepatic iron content 3 weeks after the 75 μg/kg dose. Female C57BL/10 mice are more resistant than males to the lethal effects of TCDD.

Evidence for the active renal secretion of s-pentachlorophenyl-N-acetyl-L-cysteine by female rats

Male and female rats received 50 mumoles of pentachloronitrobenzene/kg by oral intubation daily for seven days. The final excreta were hydrolysed and analysed by electron capture GLC for the presence of pentachlorobenzenethiol and tetrachloro-1,4-benzenedithiol (derived from the equivalent N-acetylcysteine conjugates). No differences were found between the sexes for faeces and bile but the urinary excretion of both thiols by females was more than 10-fold greater than males. A similar result for urine was obtained following i.p. administration of a single 20 mumoles/kg dose of S-pentachlorophenyl-N-acetyl-L-cysteine (pentachlorophenyl mercapturate); in addition co-treatment with probenecid did not greatly change excretion by the males but considerably reduced excretion by females. The sex difference in the urinary levels of pentachlorophenyl N-acetylcysteine after 40 and 100 mumoles/kg doses of pentachloronitrobenzene was confirmed by h.p.l.c. of the mercapturate and again probenecid inhibited the excretion. Analysis of urine by TLC following a dose of [14C]hexachlorobenzene (8 muCi/kg; 0.67 mumoles/kg) showed that more radioactivity was associated with the mercapturate from female rats than males. The results suggest that S-pentachlorophenyl-N-acetyl-L-cysteine, a metabolite of hexachlorobenzene and pentachloronitrobenzene, may be excreted by an active renal secretion which is particularly developed in female F344 rats.

Polycyclic aromatic hydrocarbons cause hepatic porphyria in iron-loaded C57BL/10 mice: Comparison of uroporphyrinogen decarboxylase inhibition with induction of alkoxyphenoxazone dealkylations

Multiple doses of beta-naphthoflavone to iron-loaded C57BL/10ScSn mice for 6 weeks caused inhibition of hepatic uroporphyrinogen decarboxylase and a porphyria indistinguishable from that previously only reported for polyhalogenated aromatic chemicals. beta-Naphthoflavone and other polycyclic aromatic hydrocarbon inducers of cytochrome P1-450-mediated ethoxyphenoxazone deethylation (ethoxyresorufin deethylase), benzo[a]pyrene, benz[a]anthracene, dibenz[ah]anthracene, 3-methylcholanthrene and alpha-naphthoflavone, also gave porphyria when fed. Isosafrole was inactive but by both methods phenobarbital produced a small but significant inhibition of the decarboxylase. The results demonstrate a toxic action of polycyclic aromatic hydrocarbons which probably does not involve reactive metabolites.

Sex-linked hepatic uroporphyria and the induction of cytochromes P450IA in rats caused by hexachlorobenzene and polyhalogenated biphenyls

A marked sex difference in the development of uroporphyria occurred after administration of polychlorinated and polybrominated biphenyls (PCBs and PBBs), as well as hexachlorobenzene (HCB), to F344 rats for 15 weeks. Thus the propensity of female rats to develop uroporphyria appears to be a general response to this class of halogenated chemicals. A heat-stable inhibitor(s) of liver uroporphyrinogen decarboxylase was extractable from uroporphyric livers. Although oxidation of uroporphyrinogen I to uroporphyrin I by hepatic microsomes from rats pretreated with porphyrogenic regimes of HCB and PCBs was induced, there was no correlation with the in vivo sex difference in porphyria development. Levels of total cytochrome P450 and pentoxyresorufin and benzyloxyresorufin dealkylase activities (associated with cytochrome P450IIB1) were greater in microsomes from control, HCB, PCB and PBB treated male rats than females. In contrast, ethoxyresorufin deethylase activity (associated with cytochrome P450IA1) was always significantly greater in females. These findings were confirmed by immunoblotting with polyclonal antibodies to cytochromes P450IA1, IA2 and IIB1. Immunocytochemical studies showed that, even after 30 weeks of HCB exposure, cytochromes P450IA1 and P450IA2 were still more highly induced in female liver, especially in the centrilobular region. The results are consistent with the association of cytochrome P450IA isoenzymes with uroporphyria development, although the sex difference in P450IA levels alone may not be marked enough to provide the complete explanation for the pronounced susceptibility of females to HCB.

Assay of mouse liver uroporphyrinogen decarboxylase by reverse-phase high-performance liquid chromatography

A method for the estimation of hepatic uroporphyrinogen decarboxylase activity employing reverse-phase HPLC is described. Mouse liver homogenate in 0.25 M sucrose was pretreated with a suspension of cellulose phosphate and then centrifuged to remove hemoglobin and debris. The supernatant was used as the enzyme source. Incubations were acidified, oxidized, and centrifuged only before analysis of the porphyrins formed, using a Spherisorb ODS column and a gradient solvent system constructed from methanol/lithium citrate mixtures. Coproporphyrinogen formation by BALB/c mouse liver supernatant was estimated as about 5.0 and 9.1 pmol/min/mg protein from uroporphyrinogens I and III, respectively, at 10 microM substrate concentration and pH 6.8. Decarboxylation of pentacarboxyporphyrinogens (the last step in coproporphyrinogen formation) proved to be easily measured. Coproporphyrinogen formation from pentacarboxyporphyrinogen III abd (20 microM) at pH 6.8 was about 109 pmol/min/mg protein. Pentacarboxyporphyrinogen I was not as good a substrate as III abd but was decarboxylated faster at pH 5.4 than at 6.8, and at the lower pH and at 10 microM concentration of substrate 42 pmol of coproporphyrinogen was formed/min/mg protein. These results compared favorably with those obtained by previously published procedures involving time-consuming extraction and esterification steps.

The induction of benzo[a]pyrene-3-mono-oxygenase by singlet oxygen in liver cell culture is mediated by oxidation products of histidine

The photochemical generation of excited states of oxygen by the mild illumination of culture medium containing 15 microM riboflavin results in a typical induction of benzo[a]pyrene-3-mono-oxygenase in cell lines derived from liver. However, the induction of the mono-oxygenase is not due to an excited state of oxygen directly activating the inducing mechanism inside the cell but is due to the oxidation of a component of the culture medium forming a stable inducer. The present work unequivocably shows that the component oxidised is histidine. The mild illumination of culture medium containing riboflavin therefore converts a physiological component of the medium which is not normally an inducer of the mono-oxygenase into a compound which is as effective an inducer as the classical inducer. The finding that singlet oxygen will oxidise a cell constituent into a powerful inducer is compatible with the hypothesis that excited states of oxygen and their oxidation products may play a central role in the induction of cytochrome P-450 and associated enzyme activities by many chemically unrelated inducers.

Increased inhibition of hepatic uroporphyrinogen decarboxylase by hexachlorobenzene in male rats given the oestrogenic drugs diethylstilboestrol and chlorotrianisene.

Abstract Male rats fed hexachlorobenzene (HCB) in their diet for more than 100 days showed only small changes in hepatic porphyrin concentrations and uroporphyrinogen decarboxylase activity. How-ever, when the animals received multiple doses of the oestrogenic drugs diethylstilboestrol (DES) as the dipropionate or chlorotrianisene (CTA)during the later period of feeding HCB, there was a large increase in porphyrin levels and uroporphyrinogen decarboxylase activities were severely depressed. DES and CTA did not produce porphyria in the absence of HCB. The induction of porphyria by oestrogens in male rats fed HCB, which had been proposed as a model for the latent porphyria cutanea tarda triggered by oestrogens in some humans, is therefore caused by a marked increase in the inhibition of uroporphyrinogen decarboxylase leading to the accumulation of uroporphyrin. Rats treated with DES or CTA had lower hepatic levels of HCB than those not given oestrogens, perhaps due to a greater metabolism of HCB lowering tissue concentrations. Thus the induction of porphyria in this system by oestrogens may be caused by a stimulation of HCB metabolism.

Porphyrin analysis by reversed-phase high-performance liquid chromatography: biomedical applications

Abstract Reversed-phase high-performance liquid chromatography has considerable advantages in porphyrin analysis since many of the extraction and esterification steps required for normal-phase high-performance liquid chromatography can be eliminated. This technique is of increasing importance for the diagnosis of porphyrias and in other clinical and experimental studies of porphyrins.