Gino Malvaldi

Role of the microsomal FAD-containing monooxygenase in the liver toxicity of thioacetamide S-oxide.

To evaluate the different contributions of either microsomal FAD-containing ( FADM ) or cytochrome P-450 dependent monooxygenases in the bioactivation and liver toxicity of thioacetamide-S-oxide ( TASO ) (a proximate metabolite of the liver toxin and carcinogen thioacetamide), this compound: (i) was given to rats pretreated with methimazole (a substrate and inhibitor of FADM ), SKF 525-A (an inhibitor of cytochrome P-450) and cobalt protoporphyrin IX (a synthetic porphyrin which induces a long-lasting depletion of the hepatic cytochrome P-450); and (ii) was added to liver microsomes performing oxidation of model FADM or cytochrome P-450 substrates. Whereas the prior administration of methimazole alleviated the TASO induced liver necrosis, SKF 525-A was almost ineffective. Also pretreatment with cobalt protoporphyrin IX prevented liver necrosis. However, this porphyrin derivative was found to depress both cytochrome P-450 dependent and the FADM dependent biotransformations. On the other hand, addition of TASO to liver microsomes in vitro induced changes in the kinetics of S-oxidation of thiobenzamide and of N-oxidation of dimethylaniline, whereas the O-deethylation of ethoxycoumarin was unchanged. The overall results show the necessity of TASO bioactivation by mixed-function monooxygenases for the toxic action to be apparent; at the same time, the findings suggest FADM as the system mainly involved in TASO metabolism.

Antioxidant capacity and protein oxidation in cerebrospinal fluid of amyotrophic lateral sclerosis

BackgroundThe causes of Amyotrophic Lateral Sclerosis (ALS) are unknown. A bulk of evidence supports the hypothesis that oxidative stress and mitochondrial dysfunction can be implicated in ALS pathogenesis.MethodsWe assessed, in cerebrospinal fluid (CSF) and in plasma of 49 ALS patients and 8 controls, the amount of oxidized proteins (AOPP, advanced oxidation protein products), the total antioxidant capacity (FRA, the ferric reducing ability), and, in CSF, two oxidation products, the 4-hydroxynonenal and the sum of nitrites plus nitrates.ResultsThe FRA was decreased (p = 0.003) in CSF, and AOPP were increased in both CSF (p = 0.0039) and plasma (p = 0.001) of ALS patients. The content of AOPP was differently represented in CSF of ALS clinical subsets, resulting in increase in the common and pseudopolyneuropathic forms (p < 0.001) and nearly undetectable in the bulbar form, as in controls. The sum of nitrites plus nitrates and 4-hydroxynonenal were unchanged in ALS patients compared with controls.ConclusionOur results, while confirming the occurrence of oxidative stress in ALS, indicate how its effects can be stratified and therefore implicated differently in the pathogenesis of different clinical forms of ALS.

Localization of a glutathione-dependent dehydroascorbate reductase within the central nervous system of the rat.

In this study, we describe for the first time the occurrence, within the central nervous system of the rat, of a dehydroascorbate reductase analogous to the one we recently described in the liver. Dehydroascorbate reductase plays a pivotal role in regenerating ascorbic acid from its oxidation product, dehydroascorbate. In a first set of experiments, we showed that a dehydroascorbate reductase activity is present in brain cytosol; immunoblotting analysis confirmed the presence of an immunoreactive cytosolic protein in selected brain areas. Immunotitration showed that approximately 65% of dehydroascorbate reductase activity of brain cytosol which was recovered in the ammonium sulphate fraction can be attributed to this enzyme. Using immunohistochemistry, we found that a variety of brain areas expresses the enzyme. Immunoreactivity was confined to the gray matter. Amongst the several brain regions, the cerebellum appears to be the most densely stained. The enzyme was also abundant in the hippocampus and the olfactory cortex. The lesion of norepinephrine terminals following systemic administration of DSP-4 markedly decreased immunoreactivity in the cerebellum. Apart from the possible co-localization of the enzyme with norepinephrine, the relative content of dehydroascorbate reductase in different brain regions might be crucial in conditioning regional sensitivity to free radical-induced brain damage. Given the scarcity of protective mechanisms demonstrated in the brain, the discovery of a new enzyme with antioxidant properties might represent a starting-point to increase our knowledge about the antioxidant mechanisms operating in several central nervous system disorders.

Renal involvement in feline immunodeficiency virus infection: a clinicopathological study

Renal tissues from 15 cats naturally infected with feline immunodeficiency virus (FIV) were examined histologically, immunohistochemically and ultrastructurally. Renal function and urinary proteins were also studied. Kidney abnormalities were found in 12 cats and were characterized by mesangial widening with segmental to diffuse glomerulosclerosis and presence of IgM and C3, and scanty IgG deposits in the mesangium. Tubulointerstitial lesions were also present. In 6 cats the lesions were severe enough to cause marked increase in blood urea nitrogen and creatinine, and heavy glomerular nonselective proteinuria. These findings suggest that a renal involvement is a frequent occurrence in FIV-infected cats. As the histopathological features observed were similar to those described in HIV-infected patients, FIV-infected cats may represent a valuable model for a better understanding of HIV-associated nephropathy in humans.

Chronic liver injury by thioacetamide and promotion of hepatic carcinogenesis

SummaryTo verify whether a mild, but prolonged liver injury by chemicals needing bioactivation causes both hepatic cirrhosis and the appearance of hepatocyte nodules and tumors (providing the liver has been exposed previously to initiating stimuli), diethylnitrosamine-initiated and uninitiated rats were administered thioacetamide at low dose (250 mg/l drinking water) for 6 months. Hepatocyte nodule incidence as well as changes in the drug-metabolizing system were followed at monthly intervals. In the uninitiated rats a micronodular liver cirrhosis slowly developed upon thioacetamide chronic administration; a few hepatocyte focal lesions of small size were seen from the 3rd month onward. By contrast in the diethylnitrosamine-initiated thioacetamide-treated rats the liver was macronodular because of the appearance and growth of many hepatocyte nodules; some hepatomas were also seen. During thioacetamide administration both uninitiated and diethylnitrosamine-initiated rats underwent a progressive decrease of the cytochrome P-450 liver content as well as of the activity of aminopyrine N-demethylase, ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase. On the other hand, most components of the phase II of the drug-metabolizing system were markedly enhanced. In conclusion, chronic administration of thioacetamide at low doses provided strong promoting stimuli for previously initiated hepatocytes.

Evaluation of resistance index of several anticancer agents on parental and resistant P-388 cell lines

Multidrug resistance is frequently detected in haematological malignancies and in acute leukaemias with a poor prognosis. In the last few years, several reports seem to suggest that the new anthracycline derivative idarubicin and the anthraquinone mitoxantrone have some advantages in the management of untreated or relapsed acute leukaemias compared with older anthracyclines. This could be due to a different interaction of these drugs with multidrug resistance. To evaluate this possibility, we compared the activity of doxorubicin (DOXO), epirubicin (EPI), idarubicin (IDA) and mitoxantrone (MITO) on a murine, multidrug resistant, leukaemic cell line (P-388/Dx) cultured in vitro. ID50 of IDA and MITO was in the ng range whereas that of DOXO and EPI was in the microgram(s) range. Moreover, IDA has a resistance index of 50 whereas DOXO has one of 250. Verapamil is able to almost completely abolish the resistance to IDA. Efflux experiments confirm that verapamil increases IDA intracellular concentration. IDA and MITO appear to be less involved in multidrug resistance than older anthracyclines.

The hepatotoxicity of thiobenzamide-S-oxide

Administration of thiobenzamide (TB) (0.18 mmol/100 g b.w.) to rats caused the appearance in serum and urine of a compound identified as thiobenzamide-S-oxide. When synthesized and given by oral administration, this compound induced the early appearance of liver centrilobular necrosis, impairment of glucose-6-phosphatase and aminopyrine demethylase activities, and diminished cytochrome P-450 content. Liver necrosis was suppressed by the prior administration of 20-methylcholanthrene. It is concluded that TB-S-oxide is involved, possibly as a proximate precursor, in TB-induced liver damage.

Azidothymidine in combination with 5-fluorouracil in human colorectal cell lines: In vitro synergistic cytotoxicity and dna-induced strand-breaks

The in vitro cytotoxicity of the combination of azidothymidine (AZT) and 5-fluorouracil (5-FU) against the human colorectal cancer cells SW-480, SW-620 and COLO-320DM was evaluated. The cytotoxic effects of 5-FU and AZT were determined by the assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenil)-2H-tetrazolium-5-carbo xanilide inner salt (XXT), while drug-induced DNA strand-breaks were measured using a fluorometric analysis of DNA unwinding. After an exposure of 72 h, 5-FU and AZT induced a dose-dependent cytotoxicity against each cell line. The addition of 3, 10 and 30 microM AZT to various concentrations of 5-FU, as well as the addition of 0.5, 1 and 3 microns 5-FU to various concentrations of AZT, resulted in an enhanced cytotoxic effect. Isobologram analysis and the combination index (CI) method demonstrated that the interaction between 5-FU and AZT was clearly synergistic in each cell line, except for the 30% level of effect in SW-620, where borderline synergism was observed. The evaluation of DNA strand-breaks after an exposure of 16 h to 5-FU, AZT or 5-FU + AZT demonstrated that the 5-FU + AZT combination produced the greatest DNA damage, and that this interaction was synergistic in each cell line. In conclusion, our study supports the evidence that the potential antitumour activity of AZT can be modulated by combining it with agents which inhibit thymidylate (dTMP) formation, such as 5-FU, and that the increased cytotoxicity is related to enhanced DNA damage. These findings should encourage further experimental and clinical studies of the potential use of AZT in combination with inhibitors of de novo dTMP synthesis.

Early biochemical liver changes following thiobenzamide poisoning.

Administration of thiobenzamide in a single dose (25 mg/100 g body wt by stomach tube) to male rats induced centrilobular necrosis, which became evident 10 h after the poisoning. In the meantime liver weight and water content underwent changes, glycogen was lost, triglycerides accumulated in the liver while decreasing in serum, [3H] leucine uptake in proteins was impaired and the activity of glucose-6-phosphatase and aminopyrine demethylase decreased. The activity of NADPH-cytochrome c reductase remained unchanged, whereas a reduction of the microsomal cytochrome P-450 occurred. The liver amount of reduced glutathione underwent no significant changes. Pretreatment of the animals with cobalt chloride or 20-methylcholanthrene decreased the liver damage caused by the drug. The in vitro addition of thiobenzamide to liver microsomes resulted in a spectral change. The appearance of conjugated dienes among microsomal lipids from drug-treated rats indicated for a lipoperoxidation taking place in vivo.

DETECTION OF FELINE IMMUNODEFICIENCY VIRUS P24 ANTIGEN AND P24-SPECIFIC ANTIBODIES BY MONOCLONAL ANTIBODY-BASED ASSAYS

A panel of monoclonal antibodies (mAbs) detecting distinct B-cell epitopes on p24 core viral protein of feline immunodeficiency virus (FIV) were employed to develop immunoassays to measure p24 concentration in culture and serum samples, to localize p24 in FIV-infected cells and tissues, and to detect anti-p24 antibodies in cat sera. In its optimized configuration the p24 capture assay detected as little as 0.25 ng/ml of protein. The assay was found at least as sensitive as the reverse transcriptase activity assay in FIV-infected lymphocyte cultures and proved capable of detecting p24 antigen in acid pretreated sera from a high proportion of FIV-infected cats. The mAbs were also successfully used to detect the p24 antigen in permeated FIV-infected cells by flow cytometry and in tissue sections from FIV-infected cats by immunohistochemical staining. Anti-p24 antibodies in FIV-infected cat sera were assayed by a competitive capture ELISA which readily identified occasional false positive results provided by a standard ELISA using purified whole FIV-coated wells.