BACKGROUND:The 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. METHODS =: We 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.RESULTS:The 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.CONCLUSION:Our 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.

Elevation of serum gamma-glutamyltransferase (GGT) activity is a risk factor for myocardial infarction and stroke. GGT activity can catalyze the oxidation of low-density lipoprotein (LDL), a process involved in the pathogenesis of atherosclerosis. Serum GGT is partially adsorbed onto circulating LDL, and catalytically active GGT has been found within atherosclerotic plaques, colocalizing with oxidized LDL and foam cells. We investigated the the nature of the LDL-associated GGT, the degree of correlation between total serum GGT levels and β-lipoprotein (β-LP)-associated GGT, and whether this association is altered in subjects with coronary artery disease (CAD).

Steinert's disease (myotonic dystrophy type 1; MD) is caused by a CTG trinucleotide expansion on 19q13.3. Although the pathogenic mechanism underlying multisystem involvement in MD is still unclear, a role of oxidative stress in this disease has been suggested. We investigated 39 MD patients to assess the plasma concentration of advanced oxidation protein products (AOPPs) and gamma-glutamyltransferase (GGT) and related them to clinical severity scores. Plasma AOPP levels (p=0.021), total serum GGT activity (p=0.0005) and GGT activity associated with low-density lipoprotein (p=0.00211) were significantly higher in patients than in controls. There was significant correlation between serum GGT levels and AOPPs (r=0.5831; p=0.0022). A statistically significant increase in serum GGT with age was found in MD patients (p=0.0193). Receiver operating characteristic curve analysis showed that higher AOPP levels were significantly associated with extra-muscular signs of the disease, i.e., cataracts and heart involvement (area under the curve +/- SE=0.908 +/- 0.083), but not with muscular involvement. The concomitant increment in GGT and AOPPs indicates a possible role of oxidative stress in the pathogenesis of MD type 1, while the association of increased AOPP levels with extra-muscular signs of the disease suggests that individual susceptibility to oxidative stress can modulate the extra-muscular phenotype of the disease.

Serum gamma-glutamyltransferase (GGT) is considered as a sensitive but rather nonspecific marker of hepatobiliary disease, including chronic hepatitis C virus (HCV) infection. Although its increase in HCV infection is associated with poor response to interferon-alpha (IFN-alpha) and poor prognosis, there is little knowledge of the reasons of its increase during disease.Immunohistochemistry and enzyme histochemistry were performed on fine-needle biopsies of subjects with HCV infection. GGT was detected in the lumen of bile ducts and in bile canaliculi. Furthermore, in subjects with elevated serum GGT, immunoreactive and catalytically active GGT was also detected on the sinusoidal surface of hepatocytes and diffuse cytoplasmic positivity appeared in isolated hepatocytes and hepatocellular foci. Antigen unmasking procedures showed the presence of GGT in the cytoplasm of mature and immature bile cells and of inflammatory cells. These results suggest that during chronic HCV infection there is a general enhancement of GGT activity within the liver. As the activity of several inflammatory mediators, such as leukotrienes, nitric oxide, and interleukins is modulated by GGT activity, the present findings suggest a direct relationship between serum GGT, enhanced intrahepatic GGT activity and prognosis and therapeutic outcome of chronic HCV infection.

Background. Oxidative stress plays an important role in liver ischemia/reperfusion (I/R) injury. Thus, enhancing the liver antioxidant capacity could be a promising therapeutic strategy. Ascorbate (AA) is considered the perfect antioxidant, but its therapeutic efficacy is greatly limited by its slow achievement of high intracellular levels. This might be circumvented by administering dehydroascorbate (DHA), which presents a several-fold greater uptake than AA, and undergoes rapid intracellular reduction to AA. Thus, our aim was to assess the protective role of DHA in liver I/R injury.Materials and methods. Wistar rats (200-300 g bw) were pretreated iv with different doses of AA or DRA 20 min before liver ischemia, followed by 6 h reperfusion. Liver damage was assessed by biochemical and morphological indices.Results. DRA pretreatment induced a rapid increase in liver ascorbate levels, significantly higher than findings for AA, without any significant reduction in glutathione levels. Liver damage during I/R in controls showed significant increases in serum transaminases and hepatic thiobarbituric acid reactive substances with alterations of liver morphology. DHA administration induced a clear, significant protection against I/R injury, whereas liver damage was only moderately prevented by AA.Conclusions. DRA might represent a simple, effective therapeutic option to prevent liver damage associated with ischemia/reperfusion. (C) 2004 Elsevier Inc. All rights reserved.

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.

Because abnormalities in redox balance cluster in type I diabetes families and the intracellular thiol redox status seems to modulate immune function, we aimed to investigate the relationship between oxidative stress and immunological features. We measured oxidative markers, serum proinflammatory cytokines, soluble cytokine receptors and subsets of peripheral blood lymphocytes (by varying combinations of CD4, CD8, CD23 or low-affinity IgE receptor, and CD25 or IL-2 receptor) from 38 type I patients, 76 low-risk (i.e. without underlying islet autoimmunity) non-diabetic first-degree relatives of diabetic patients, and 95 healthy subjects. In type I diabetes families, protein and lipid oxidation was confirmed by the presence of reduced sulphhydryl groups, increased advanced oxidation protein products, and increased plasma and erythrocyte malondialdehyde. Relatives had decreased counts of monocytes, of cells co-expressing CD23 and CD25 and of CD25(+) cells in peripheral blood. Patients with TIDM had similar defects and, in addition, showed decreased counts of peripheral CD4(+)CD8(+) lymphocytes and increased serum levels of soluble receptors for interleukin (IL)-6 and IL-2. Abnormal indicators of oxidative stress were related in part to immune abnormalities. In the whole study group, we found a correlation (multiple R 0.5, P < 0.001) of CD23(+)CD25(+) cells with blood counts of monocytes, CD4(+)CD8(+) cells, CD25(+) cells, basal haemolysis and plasma levels of thiols. In type I diabetics, anti-GAD65 antibody levels were associated (multiple R 0.6, P = 0.01) positively with sIL-6R, negatively with duration of diabetes and CD23(+)CD25(+) counts; plasma creatinine correlated positively (multiple R 0.6, P < 0.001) with both sIL-2R and tumour necrosis factor (TNF)-alpha concentration. Our study reports the first evidence that the oxidative stress observed in type I families is related to immunological hallmarks (decreased peripheral numbers of monocytes as well as cells bearing a CD4(+)CD8(+), CD23(+)CD25(+) and CD25(+) phenotype) from which the involvement of some immunoregulatory mechanisms could be suspected. It remains to be elucidated the course of events culminating in the loss of physiological immune homeostasis and disease pathology.

Ascorbic acid (AA) is an important factor of defence against oxidative stress. AA is maintained in the reduced functional form by glutathione (GSH)-dependent dehydroascorbate (DHA) reducing enzymes, including the cytosolic glutaredoxin, the microsomal protein disulphide isomerase, and a DHA reductase of 31 kDa, hereafter referred to as DHAR, purified from rat liver cytosol and human red cells. As these mechanisms have rarely been studied in parasites, we looked for the possible presence of this 31 kDa protein in Trichinella spiralis L(1) larvae. Biochemical data, immunoblot analysis and immunohistochemical studies suggested the absence of this protein within parasites at this stage. However, they possess a low DHA reducing ability, which is probably due to the presence of glutaredoxin. On the other hand, immunohistochemical studies performed in histological sections of muscle tissue from Trichinella-infected animals showed an increase in DHAR in the nurse cell (NC) of T. spiralis- and Trichinella britovi-infected animals, compared with the surrounding muscle fibres. This result was confirmed by immunoblot analysis, whereas no such increase was observed in Trichinella pseudospiralis-infected animals. In the modified skeletal muscle cell also haeme oxygenase 1 increased, as well as lipoperoxidised proteins. Both findings suggest an oxidative stress of the NC, which might be related to the intense inflammatory reaction which surrounds the NC-parasite complex. Another possibility to explain the increase in DHAR could be that the NC needs to recycle a substantial amount of AA to synthesise the collagen capsule.

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.

Background. Experimental studies have demonstrated that 5-fluorouracil (5-FU) enhances zidovudine (AZT)-induced DNA strand breaks and cytotoxicity. Phase I studies have demonstrated that the maximum tolerable dose (MTD) of AZT is 8000 mg/sqm when administered i.v. over two hours after weekly 5-FU + 1-leucovorin (LV), and that this combination has promising antitumor activity. The purpose of this study was therefore to evaluate the antitumor activity of weekly bolus 5-FU + LV + AZT, administered al its MTD, and to determine whether 5-FU enhances AZT-induced DNA strand breaks in blood nuclear cells.Patients and methods. Twenty-nine chemotherapy-naive metastatic colorectal cancer patients with measurable disease entered the study to evaluate the activity of a weekly 5-FU 500 mg/m(2) i.v. bolus + LV 250 mg/m(2) i.v. two-hour infusion + AZT 8000 mg/m(2) i.v. two-hour infusion. In 10 different patients, who during three different weeks received 5-FU + LV, AZT and 5-FU + LV + AZT, DNA strand breaks in blood nuclear cells were determined by a fluorescent analysis of DNA unwinding.Results: Treatment was generally well tolerated and WHO grades III-IV toxicities, consisting mostly of diarrhea (17%), were uncommon. One patient died of severe diarrhea with consequent hypokalemia and cardiac arrhythmia. All patients were considered evaluable for response, and 3 (10%) complete and 10 (35%) partial responses were observed, for an objective response rate of 45% (95% confidence limit interval 26%-64%). Both 5-FU + LV and AZT decreased the percentage of double stranded DNA in nuclear blood cells. The greatest effect was observed with 5-FU + LV + AZT which reduced the percentage of double stranded DNA to 50% and 36% after 24 and 48 hours, respectively, and this interaction between 5-FU + LV and AZT was found to be cumulative.Conclusions. These studies demonstrate that the present dose and schedule of AZT in combination with 5-FU + LV has significant activity in metastatic colorectal cancer and that the combination of 5-FU + LV with AZT increases the amount of DNA damage. Therefore, AZT in combination with 5-FU + LV warrants further study in colorectal cancer.

S-Adenosyl-L-methionine (SAM) is a strong chemopreventive agent of rat liver carcinogenesis. Examination was made to determine whether inhibition by SAM of the development of preneoplastic liver lesions persists to SAM withdrawal in diethylnitrosamine-initiated F344 rats promoted with thiobenzamide (TB). The rats were subjected, 2 weeks after initiation, to 5 weeks feeding with a 0.1% TB diet followed by a TB-free diet for 6 weeks and then a second TB treatment for 3 weeks. SAM (384 micromol/kg/day) was injected i.m. during the first TB cycle (treatment A) or for 6 weeks after the first TB cycle (treatment B). Many gamma-glutamyltranspeptidase (GGT)-positive lesions developed in initiated rats after the first TB cycle. They decreased in number after TB withdrawal, while partial recovery of lesion number and a great increase in volume occurred after the second TB cycle. Liver ornithine decarboxylase (ODC) activity and c-myc and c-Ha-ras mRNAs increased during the TB cycles and returned to normal liver values after TB withdrawal. Number and size of GGT-positive lesions, DNA synthesis of GGT-positive cells, liver ODC activity and c-myc and c-Ha-ras mRNA levels decreased as a consequence of SAM treatment A. The recovery of these parameters, induced by a second TB cycle in rats not treated with SAM, was prevented by SAM treatment B. These results suggest that SAM causes a persistent decrease in growth capacity of preneoplastic liver lesions in rats subjected to a diethylnitrosamine/TB protocol.

A novel GSH-dependent dehydroascorbate (DHA) reductase from rat liver cytosol has been recently purified and partially characterized in our laboratory. A further characterization study has been carried out in order to determine intracellular and tissue distribution of the enzyme. A modified purification method, yielding a threefold increase in enzyme activity recovery, has been used. Polyclonal antibodies were obtained in rabbits and specific anti-DHA reductase IgG were purified by affinity chromatography employing the homogeneous enzyme as ligand. Immunoblotting analysis of subcellular fractions showed the exclusively cytosolic location of the enzyme. Immunotitration experiments, performed in order to determine the percentage of cytosolic DHA reductase activity ascribable to our enzyme, revealed that purified enzyme activity was completely titrable, while only 70% of DHA reducing activity was titrable in liver cytosol preparation. When immunoblotting analysis was employed to determine tissue distribution of the enzyme, liver, intestinal mucosa, kidney, adrenals, submaxillary gland, testis, and pancreas appeared most endowed with the enzyme, and lower levels were observed in all the other tissues examined. Immunohistochemical studies showed clear zonal distributions in kidney and intestinal tract and overall homogeneous patterns in the other tissues.

This chapter discusses substrate interactions of protein prenyltransferases. The posttranslational modification of proteins by isoprenoids facilitates protein–membrane and protein–protein interactions. This modification is common in a variety of proteins. It is estimated that as much as 0.5% of mammalian brain proteins are prenylated. Among the known prenylated proteins are most of the small guanosine triphosphate (GTP)-binding proteins, the γ subunits of trimeric G proteins, and nuclear lamins. Protein prenylation is likely to affect a wide variety of cellular functions. Prenylation of proteins is catalyzed by protein prenyltransferases. In the reaction a thioether bond is formed between the sulfhydryl group of a cysteine residue, located near the carboxy terminus of the acceptor protein, and either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid. The nature of the prenyl modification is highly dependent on the specificity of the prenyltransferases. The three protein prenyltransferases isolated are (1) farnesyltransferase (FTase), (2) geranylgeranyltransferase I (GGTase I), and (3) geranylgeranyltransferase II (GGTase II).The determination of farnesyl pyrophosphate binding assay using nonisolated enzyme-[3H] farnesyl pyrophosphate complex is described in the chapter.

To the Editor: Glutathione (GSH) is the most abundant acid-soluble thiol in biological fluids, where it is involved as a cofactor in several functions, ranging from glycolysis to regulation of the cell cycle (1,2). As far as detoxication of reactive intermediates is concerned, GSH may act as a nucleophile, forming conjugates, or as reductant, being oxidized to glutathione disulfide (GSSG) (3). Most of these reactions, which may protect cells from the harmful effects of oxidative stress, are catalyzed by glutathione S-transferases (GSH-T) and GSH peroxidases (GSH-Px), whereas GSH-reductase (GSH-R) is the main enzyme involved in the reduction of GSSG (3-5). A role for GSH in the progression of HIV-induced pathology has been inferred from the repeated findings of decreased GSH levels in plasma, lung epithelial lining fluid, erythrocytes, lymphocytes, and T-cell subsets in HIV-infected patients (6). In view of the relevant redox buffering capability of GSH, decreased GSH concentration may suggest that an increased oxidative stress could be relevant for the pathogenesis of virus-induced disease as well as immunosuppression because low GSH levels inhibit many T- and B-cell functions, including the effector phase of cytotoxic T-cell responses and the IL-2-dependent cell activities (7). Despite the elevated levels of lipid peroxidation byproducts, such as malondialdehyde, found in HIV-infected patients, the nature as well as the biological meaning of the changes involving the antioxidant systems remain quite controversial because some of the contrasting findings reported so far could depend on technical difficulties to obtain and express comparable data (8-10). To investigate whether changes in the GSH-dependent antioxidant system also may develop during feline immunodeficiency virus (FIV) infection and FIV-induced pathology, both the concentration of GSH and the activity of some enzymes involved in GSH metabolism were assessed and compared in naturally and experimentally infected cats as well as in FIV-infected cultures of the Crandell feline kidney (CrFK) cell line. Naturally infected field cats and their controls were selected (with the owners' consent) from those referred to the Animal Clinic of the University of Pisa and found positive for FIV antibody in serum by a commercial test (Cite-combo FIV-FeLV, Agritech System, Portland, ME, U.S.A.); at sampling, none of the FIV-positive cats had symptoms compatible with overt feline AIDS-related complex. The FIV-seronegative controls were healthy cats referred to the clinic for unrelated problems and matched insofar as possible to the infected cats for breed, age, gender, and weight. None of the animals from either group was positive for feline leukemia virus antigen. Specific pathogen-free (SPF) cats were purchased from Iffa Credo (L'Asbrege, France) and housed individually in climatized animal facility under EC law conditions. Experimental FIV infection was performed when the cats were aged 6 months with the Pisa-M2 isolate; SPF-FIV+ cats were sampled 28 months after infection, when they displayed an evident loss of CD4+ lymphocytes. Uninfected control animals were age- and sex-matched SPF cats maintained under identical conditions but in a separate quarter. Blood specimens were collected, with the cats under slight anesthesia, in tubes containing EDTA from the jugular vein. Peripheral blood mononuclear cells (PBMC) were harvested by density gradient centrifugation on Histopaque (Sigma, St. Louis, MO, U.S.A.); after washing, counting, and CD4+ enumeration by the fluorescence-activated cell sorter (FACS) (11), they were pelleted for biochemical determinations. GSH and GSSG content was measured by highperformance liquid chromatography (HPLC) assay using 2,4-dinitro-fluorobenzene according to Reed et al. (12). The activity of GSH-T, GSH-Px, and GS-R as well as the protein concentration within each sample were measured and expressed as detailed in Pratesi et al. (13). Differences between experimental groups were calculated by either one-way analysis of variance or Student's t test using a p value of 0.05 as significance level. The main results obtained by measuring GSH/GSSG concentration as well as the activity of the relevant enzymes involved in the consumption and recycling of this acid-soluble thiol within PBMC from FIV-positive cats are shown in Table 1. As far as the field cats are concerned, no changes were seen in either GSH or GSSG contents of the PBMC; also, the activities of GSH-T and GSH-R were unchanged. The only difference between controls and FIV-infected animals was a borderline decrease (-39.9%; p = 0.049) of the activity of GS-R; accordingly, the capability of cell to recycle GSSG to GSH should be slightly decreased; on the other hand, the small increase in intracellular GSSG observed in FIV-infected field cats when expressed on a “per cell” basis (1.1 ± 0.6 nmol GSSG/107 cells versus 0.6 ± 0.3 of the controls) did not reach statistical significance. It should be noted that the time of seroconversion of the field cats was unknown; furthermore, the cats were in good health as shown from the absence of clinical symptoms and CD4+ count changes. Because, in addition to the FIV infection, FIV-induced pathology also could play a role in modifying the overall GSH metabolism, all relevant determinations of GSH status were repeated on SPF cats experimentally infected with FIV Pisa-M2 strain from 28 months. At this time, a significant decrease of both CD4+ count and of GSH content of the PBMC was observed without relevant changes of the plasma concentration of GSH (2.6 ± 0.3 nmol/ml in FIV-positive cats versus 2.7 ± 0.5 in controls). The GSSG content of PBMC was unchanged with respect to controls, whereas GSSG plasma concentration was somewhat increased (2.9 ± 0.9 nmol/ml in FIV-positive cats versus 1.9 ± 0.1 in the controls, p < 0.05). Among the glutathione-related enzymes, GSH-T was not affected by FIV infection, whereas both GSH-Px and GSH-R underwent a significant decrease with respect to controls. Because the effects of FIV infection on the GSH-based antioxidant systems seem enhanced from the FIV-induced cell damage, GSH content as well as GSH-related enzyme activity were assayed in FIV permissive CrFK cells at varying times after FIV infection, that is, in the absence as well as in the presence of FIV-induced cytopathology (namely, syncytia). The Pisa-M2 FIV isolate was propagated in CrFK cells adapted to grow in the presence of 0.5% fetal calf serum and highly susceptible to syncytium formation by this virus (14). Biochemical determinations were performed 2, 4, and 6 days after infection. The decrease of cell GSH (10.9 ± 1.7 nmol/mg protein versus 15.5 ± 1.9 of the controls, p < 0.05) and of GSH-Px activity (31 ± 6.1 nmol NADPH oxidized/min/mg protein versus 14.5 ± 3.6 of the controls, p < 0.05) reached its maximum 6 days after infection, when the number of FIV-induced syncitia was very high (14); a decrease of GSH-R was evident from 2 days postinfection (15.1 ± 2.7 nmol NADPH oxidized/min/mg protein versus 29 ± 6.2 of controls, p < 0.05), whereas GSH-T always showed no changes. To summarize the results we obtained, we point out that FIV infection is followed by multiple alterations of GSH metabolism. A major determinant of these changes, which are consistent with an overall decrease in GSH availability within the cell, however, seems to be FIV-induced cytopathology rather than FIV infection as such. Data obtained from FIV-infected cats are consistent with similar findings obtained from HIV-infected patients and suggest the FIV model as being quite suitable for a deeper insight into the mechanism(s) of HIV-induced pathology in regard to oxidant stress-related disturbances. The FIV model lends itself to direct experimental testing of the hypotheses under investigation and could help both to clarify some of the many conflicting results so far obtained in HIV-infected patients and to evaluate the possibility of counteracting disease progression by combining antiviral and antioxidant therapy. Acknowledgment: This work was supported by grants from Ministero della Sanità, Istituto Superiore di Sanità, “Progetto Allestimento Modelli Animali per l'AIDS” and Ministero della Università e della Ricerca Scientifica, Roma, Italy. *Aldo Paolicchi; *Patrizia Tonarelli; *Solange Silva; ‡Patrizia Bandecchi; *†Gino Malvaldi *Department of Biomedicine; †Retrovirus Center; ‡Department of Animal Pathology; University of Pisa; Pisa, Italy

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.

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 μg 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.

Flow cytometry has been employed to study NK cell cytotoxic activity in cats infected with feline immunodeficiency virus. The results show that animals infected for 12 months or more have decreased levels of NK cell cytotoxic activity in their blood. The impairment could not be overcome by in vitro treatment of effector cells with interleukin 2. Additional results suggest that the NK cells of infected cats are defective, in that they are still able to bind to target cells but have a reduced ability to kill them.