New familial cases of karyomegalic interstitial nephritis with mutations in the FAN1 gene.

BACKGROUND: Karyomegalic interstitial nephritis (KIN) is a rare disease entity first described by Burry in 1974. The term KIN was introduced by Mihatsch et al. in 1979. KIN is characterized by chronic tubulointerstitial nephritis associated with enlarged tubular epithelial cell nuclei, which leads to a progressive decline of renal function. The prevalence of this disease is less than 1% of all biopsies, and its pathogenesis is unclear. KIN results from mutations in FAN1 (FANCD2/FANCI-Associated Nuclease 1), a gene involved in the DNA damage response pathway, particularly in the kidney. In this study, we report two Tunisian consanguineous families with KIN caused by mutations in the FAN1 gene. METHODS: Direct sequencing of the coding regions and flanking intronic sequences of the FAN1 gene was performed in three affected members. Three prediction programs (Polyphen-2 software, SIFT, and MutationTaster) were used to predict the functional effect of the detected variations. RESULTS: Two causative frameshift variants in the FAN1 gene were identified in each family: The previously described frameshift mutation c.2616delA (p.Asp873ThrfsTer17) and a novel mutation c.2603delT (p.Leu868ArgfsTer22) classified as "pathogenic" according to the American College of Medical Genetics and Genomics (ACMG) guidelines. CONCLUSION: To our best knowledge, this is the first Tunisian study involving familial cases of KIN with mutations in the FAN1 gene. We hypothesize that these findings can expand the mutational spectrum of KIN and provide valuable information on the genetic cause of KIN.

The in vitro effects of zearalenone and T-2 toxins on Vero cells.

The aim of the present study was to investigate in vitro, whether cytolethality and oxidative damage is enhanced by combination of both mycotoxins as compared to their individual effect. In our paper, we applied a tiered in vitro experimental approach in order to predict the possible health risk effects of two interactive fusarial toxins. Considering the concomitant production of zearalenone (ZEN) and T-2 toxin, it is very likely that humans and animals are always exposed to the mixture rather than to individual compounds. Our results clearly showed that cultured renal cells respond to individual (ZEN) or T-2 toxin exposure by a moderate inhibition of cell proliferation, respectively. However, when combined, they exert a more significant decrease in cell viability. Similar results were found for the investigated oxidative status endpoints. When combined, ZEN and T-2 toxin increased ROS production and heat shock protein (Hsp) 70 expression as compared to the effect of each mycotoxin taken alone. We can conclude that the mixture of ZEN and T-2 toxin increased their toxic effects. The health risk is heightened by the interactions between co-occurring mycotoxins.

Cytochrome P450 (CYP3A4*18) and glutathione-S-transferase (GSTP1) polymorphisms in a healthy Tunisian population.

AIM: Human cytochrome P450 3A and glutathione S-transferase (GST) enzymes evolved to catalyze the metabolism of numerous common therapy drugs and endogenous molecules. Members of the CYP3A are the majority expressed in human liver and intestine. The genetic factors play an important role in the interindividual variability in CYP3A and GST activity. Detection of CYP3A4 and GST variant alleles and knowledge about their allelic frequency in specific ethnic groups are important to lead to individualized drug dosing and improved therapeutics. METHODS: We determined the allelic frequency of the CYP3A4*18 and GSTP1 in a group of 138 healthy Tunisian subjects using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. It is based on a specific PCR product cut by restriction endonucleases. This method offers the advantage of cutting DNA by the appropriate restriction enzyme at the correct mutation site hence enhancing its reliability. Electrophoretic separation demonstrates the presence (or absence) of restriction sites. RESULTS: In the group of 138 unrelated individuals, the frequencies of the CYP3A4*18 and GSTP1 variant allele in this Tunisian population were 0.02 and 0.01, respectively. CONCLUSIONS: The present study describes polymorphisms of Cyp3A4 and GST among Tunisian subjects. We developed a simple assay for the detection of the CYP3A4*18 and GSTP1 polymorphisms and we compared our allelic frequencies to other populations. No significant difference was obtained. This study provides the first analysis of CYP3A4*18 and GSTP1 mutant allele frequencies in the Tunisian population.

Ochratoxin A induces CYP3A4, 2B6, 3A5, 2C9, 1A1, and CYP1A2 gene expression in primary cultured human hepatocytes: a possible activation of nuclear receptors.

Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic, and immunotoxic to several species of animals and to cause kidney and liver tumors in mice and rats. Biotransformation of OTA has not been entirely elucidated. Several metabolites have been characterized in vitro and/or in vivo, whereas other metabolites remain to be characterized. At present, data available regarding OTA metabolism and cytochrome inductions concern only rodents or in vitro systems. The aim of the present study was to explore the effect of OTA on mRNA expression of some cytochromes known to be regulated by pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR), using primary cultures of human hepatocytes. Our results showed that OTA reduced hepatocyte viability in a dose-dependent manner. Using quantitative real-time reverse-transcription polymerase chain reaction, our study showed that treatment of primary cultured human hepatocytes with noncytotoxic increasing concentrations of OTA for 24 hours caused a significant upregulation of CYP3A4, CYP2B6, and, to a lesser extent, CYP3A5 and CYP2C9. PXR mRNA expression increased in only 1 treated liver, whereas CAR mRNA expression was not affected. OTA was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that OTA could activate PXR and AhR; however, further investigations are needed to confirm nuclear-receptor activation by OTA.

Presence of ochratoxin A in Tunisian blood nephropathy patients. Exposure level to OTA.

Ochratoxin A (OTA) produced by Aspergillus and Penicillium genera contaminates cereals and different food compounds. OTA presents a wide range of toxic effects, especially nephrotoxicity. It is also considered to be the main causal agent of Balkan Endemic Nephropathy (BEN) which is similar to the Chronic Interstitial Nephropathy with unknown aetiology seen in Tunisia. In this study, we attempted to confirm the relationship between OTA blood levels and the development of renal pathology. Hence, serum OTA levels were measured in several groups of patients having different renal diseases: a group presenting Chronic Interstitial Nephropathy (CIN) with unknown aetiology, a group presenting Chronic Interstitial Nephropathy (CIN) with known aetiology, a group presenting Chronic Glomerular Nephropathy (CGN), and a group presenting Chronic Vascular Nephropathy (CVN). Each group was compared to a healthy control group. In the healthy group, 49% of individuals showed OTA concentrations ranging from 1.7 to 8.5 ng/ml, with a mean value of 3.3±1.5 ng/ml. However, among nephropathic patients, the group with CIN of unknown aetiology showed the highest incidence (76%), ranging from 1.8 to 65 ng/ml with a mean value of 18±7 ng/ml. Even in the healthy group, the calculated Daily Intake (DI) ranged from 5.0 to 24.9 ng/kgb.w./day when compared to the recommended DI by the scientific committee on foods of 5 ng/kgb.w./day, indicating a high degree of exposure to OTA in the Tunisian population. Our study confirms the involvement of this nephrotoxic mycotoxin, present at high blood levels in the Tunisian population, in the outcome of this particular human nephropathy (CIN with unknown aetiology) which is similar to BEN.

Cytotoxic effects exerted by polyarylsulfone dialyser membranes depend on different sterilization processes.

Polyarylsulfone group is one of the most important polymeric materials used in the biomedical field, due to its excellent properties, such as good thermal, chemical, and mechanical stability. There are three important polyarylsulfone polymers, all of which have excellent electrical properties: polysulfone (PSu), polyarylsulfone (PAS) and polyarylethersulfone (PAES). All these polymers have excellent creep, radiation and high temperature resistance. In this study, we aimed to determine the effect of three sterilization processes (steam, ethylene oxide and gamma rays) on cytotoxicity of polyarylsulfone dialysis membranes. Ten long-term dialysis patients and ten age-matched healthy controls were enrolled in our study. We analysed (1) serum effect on cultured endothelial cell viability using MTT assay and (2) lipid peroxidation assessed by serum malondialdehyde (MDA) formation at the beginning (T0), the middle (T2) and the end (T4) of haemodialysis (HD) session. Our results clearly showed that steam-sterilized membranes improve endothelial cell viability when compared to ethylene oxide or gamma rays-sterilized ones. Moreover, there is a increased generation of MDA in patients sera during HD session. The serum MDA concentration was about 3, 6 and 10 times higher, respectively, for steam, ethylene oxide and gamma rays sterilization procedures when compared to the MDA amount in healthy subject sera. We concluded that using steam instead of ethylene oxide or gamma rays for sterilization may improve the biocompatibility of polyarylsulfone membranes.

Ochratoxin A levels in spices and dried nuts consumed in Tunisia.

A total of 112 samples of spices (24 caraway, 20 coriander, 25 curcuma, 20 black pepper and 23 red pepper) and 110 samples of dried nuts (44 almonds, 42 peanuts and 24 pistachio) purchased from popular markets in 24 regions of Tunisia were analyzed for ochratoxin A (OTA) by fluorescence HPLC. The average levels of contamination of OTA found in spice samples were 244, 206, 290, 274 and 203 µg/kg, respectively, for caraway, coriander, curcuma, black pepper and red pepper. Concerning dried nut samples, the average levels were 61, 60 and 89 µg/kg, respectively, for almonds, peanuts and pistachio. Contamination levels were higher than the usual norms (10.0 OTA µg/kg) established by the European Commission in 2005 . This survey is the first to be carried out on the natural occurrence of OTA in the main spices and dried nuts consumed by the Tunisian population.