Rossana Jorquera

Cyclin B-dependent kinase and caspase-1 activation precedes mitochondrial dysfunction in fumarylacetoacetate-induced apoptosis

Hereditary tyrosinemia type I is the most severe metabolic disease of the tyrosine catabolic pathway mainly affecting the liver. It is caused by deficiency of fumarylacetoacetate hydrolase, which prevents degradation of the toxic metabolite fumarylacetoacetate (FAA). We report here that FAA induces common effects (i.e., cell cycle arrest and apoptosis) in both human (HepG2) and rodent (Chinese hamster V79) cells, effects that seem to be temporally related. Both the antiproliferative and apoptosis‐inducing activities of FAA are dose dependent and enhanced by glutathione (GSH) depletion with L‐buthionine‐(S,R)‐sulfoximine (BSO). Short treatment (2 h) with 35 µM FAA/+BSO or 100 µM FAA/—BSO induced a transient cell cycle arrest at the G2/M transition (20% and 37%, respectively) 24 h post‐treatment. In cells treated with 100 µM FAA/–BSO, an inactivation, followed by a rapid over‐induction of cyclin B‐dependent kinase occurred, which peaked 24 h post‐treatment. Maximum levels of caspase‐1 and caspase‐3 activation were detected at 3 h and 32 h, respectively, whereas release of mitochondrial cytochrome c was maximal at 24–32 h post‐treatment. The G2/M peak declined 24 h later, concomitantly with the appearance of a sub‐G1, apoptotic population showing typical nucleosomal‐sized DNA fragmentation and reduced mitochondrial transmembrane potential (Δψm). These events were prevented by the general caspase inhibitor z‐VAD‐fmk, whereas G2/M arrest and subsequent apoptosis were abolished by GSH‐monoethyl‐ester or N‐acetylcysteine. Other tyrosine metabolites, maleylacetoacetate and succinylacetone, had no antiproliferative effects and induced only very low levels of apoptosis. These results suggest a modulator role of GSH in FAA‐induced cell cycle disturbance and apoptosis where activation of cyclin B‐dependent kinase and caspase‐1 are early events preceding mitochondrial cytochrome c release, caspase‐3 activation, and Δψm loss.—Jorquera, R., Tanguay, R. M. Cyclin B‐dependent kinase and caspase‐1 activation precedes mitochondrial dysfunction in fumarylacetoacetate‐induced apoptosis. FASEB J. 13, 2284–2298 (1999)

Involvement of Endoplasmic Reticulum Stress in Hereditary Tyrosinemia Type I

Hereditary tyrosinemia type I (HTI) is the most severe disease of the tyrosine degradation pathway. HTI is caused by a deficiency of fumarylacetoacetate hydrolase (FAH), the enzyme responsible for the hydrolysis of fumarylacetoacetate (FAA). As a result, there is an accumulation of metabolites such as maleylacetoacetate, succinylacetone, and FAA. The latter was shown to display mutagenic, cytostatic, and apoptogenic activities and to cause chromosomal instability. Herein, we demonstrate that FAA also causes a cellular insult leading to the endoplasmic reticulum (ER) stress signaling. Treatment of V79 Chinese hamster lung cells with an apoptogenic dose of FAA (100 μm) causes an early induction of the ER resident chaperone GRP78/BiP and a simultaneous phosphorylation of the eIF2α. FAA treatment also causes a subsequent induction of the proapoptotic CHOP (CEBP homologous protein) transcription factor as well as a late activation of caspase-12. Data obtained from fah–/– mice taken off the therapeutic 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione drug are similar. However, in this mouse model, there is also an increase in proteasome activity indicative of ER-associated degradation. This difference observed between the two models may be due to the fact that the murine model measures the effects of all metabolites accumulating in hereditary tyrosinemia type I as opposed to the cellular model that only measures the effects of exogenous FAA.

Expression of the cytochrome P4502e and 2B gene families in the lungs and livers of nonpregnant, pregnant, and fetal hamsters

Members of the cytochrome P4502E and 2B gene families have been implicated in the activation of nitrosamines to reactive species capable of binding to cellular macromolecules and initiating tumor formation in various rodent species. This study was initiated to determine the relative prevalence of these isozymes and their response to ethanol during pregnancy and late gestation. Nonpregnant and pregnant hamsters were given a 10% ethanol solution in their drinking water for 10 days (gestation days 5-15) prior to being killed. RNA blot analysis of liver and lung tissue from nonpregnant, pregnant, and fetal hamsters demonstrated tissue-specific expression of CYP2E and 2B in adult and fetal animals. The levels of RNA expression of both P450s in fetal hamsters were less than 30% of nonpregnant adult values. In pregnant hamsters, the hepatic levels of CYP2E and 2B RNAs were decreased compared to nonpregnant animals. In contrast, the pulmonary levels of CYP2B RNA were increased in pregnant versus non-pregnant hamsters, while no effect of pregnancy on the levels of CYP2E RNA was seen. Although rats contain a single CYP2E1 gene transcript. Northern analysis demonstrated the presence of 1.8 and 2.8 kb bands in both liver and lung tissue of the hamster. Pretreatment with ethanol had little effect on the levels of either P450 RNA species in the lungs or livers of nonpregnant, pregnant, and fetal hamsters. These results demonstrate differences in the levels of expression of members of the CYP2E and 2B gene families during pregnancy and late gestation compared to nonpregnant adult hamsters. Fetal animals, like the adults, apparently respond to ethanol treatment by altering the levels of these P450 isozymes at the post-transcriptional level.

Transplacental carcinogenicity of low doses of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone administered subcutaneously or intratracheally to hamsters

Our previous studies have demonstrated that doses of 300–50 mg/kg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) injected subcutaneously into pregnant hamsters cause a 44% incidence of respiratorytract tumors in the offspring. In this study, we have extended the assay of the carcinogenic potency of NNK to doses ranging from 50 mg to 0.05 mg/kg body weight and to a second route of administration, intratracheal instillation, which is more relevant to inhalation of tobacco smoke by pregnant women. Among the offspring whose mothers had been injected subcutaneously with NNK (20–1 mg/kg), the total tumor incidence (57.2%–16.7%) decreased with decreasing dose levels. After intratracheal instillation of 50–0.05 mg/kg NNK the overall incidence varied from 28.6% to 50% but no dose response was observed. The main target organs were the adrenal glands (Phaechromocytomas) and nasal cavities (adenocarcinomas of the olfactory region). A low incidence of ductular adenomas of the pancreas was observed with low doses of NNK instilled intratracheally. These results demonstrate that NNK, at doses that are comparable to the cumulative exposure during a 9-month period in women, is a potent transplacental carcinogen in hamsters.

Effects of age and ethanol on DNA single-strand breaks and toxicity induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or n-nitrosodimethylamine in hamster and rat liver.

The effects of age and ethanol exposure on liver DNA single-strand breaks (SSB) and liver cell injury induced in hamsters and rats by a single equimolar dose (0.39 mmol/kg) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or N-nitrosodimethylamine (NDMA) were investigated. NNK induced more DNA SSB (by 10-50%) than NDMA in the liver of adult hamsters and rats, but similar differences were not observed in newborn animals. Nitrosamine-induced hepatic DNA damages was compared in newborn and adult animals. While newborn hamsters were less sensitive to NNK-induced DNA damage than adult hamsters, newborn rats were more sensitive to NDMA-induced DNA damage than adult rats. In utero ethanol exposure did not alter significantly the induction of hepatic DNA SSB by NNK or NDMA compared to newborn hamsters and rats. Interestingly, species differences in the extents of NNK-induced hepatic DNA SSB and toxicity were observed in ethanol-consuming adult hamsters and rats. Ethanol treatment of hamsters caused a significant reduction (by 35%) of the frequency of hepatic DNA SSB and a 3.5-fold enhancement of hepatotoxicity induced by NNK.

Chapter 40 – Hepatorenal Tyrosinemia

Publisher Summary Hereditary tyrosinemia type I (HTI), also referred to as hepatorenal tyrosinemia, is the most severe metabolic disorder of the tyrosine degradation pathway. HTI is caused by a deficiency in fumarylacetoacetate hydrolase (FAH), the last enzyme of the pathway, which catalyzes the conversion of fumarylacetoacetate (FAA) in fumarate and acetoacetate. HTI presents into two main clinical types, acute and chronic. The acute form, with early onset at birth or soon after, is mainly characterized by severe liver failure associated with cirrhosis, hepato- and splenomegaly, abnormal blood coagulation and hypoglycemia leading to death in the first months of life. Renal tubular dysfunctions such as Fanconi syndrome and rickets have also been considered signs of HTI. In the chronic form, onset is insidious and progress is less aggressive. However, renal manifestations, mainly proximal tubulopathy, are prominent. Patients show impaired renal tubular reabsorptive function leading to Fanconi syndrome characterized by renal tubular acidosis, generalized aminoaciduria, hypophosphatemic vitamin D-resistant rickets and growth retardation. In vivo reversion of inherited somatic mutations has been reported in a number of inherited diseases, such as adenosine deaminase deficient severe combined immuno deficiency, Wiskott-Aldrich and Bloom syndromes, epidermolysis bullosa, Fanconi anemia, and HTI. It is in HTI, however, that reversion has been shown to occur most frequently. A mosaic pattern of FAH expression was observed in hepatic regenerative nodules of ›85% of studied patients. DNA analysis performed on FAH immunopositive liver nodules showed correction of the HTI-causing mutation in one allele. The treatment currently used in HTI patients is administration of the drug (2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3 cyclohexanedione) (NTBC) combined with a low-protein diet.