Hepatoceliular carcinoma (HCC) represents a major cause of mortality in certain areas ofthe world. Contamination offood with the mycotoxin aflatoxin B1 (AFB1) has been implicated as an etiological factor in certain regions of eastern Asia and subSaharan Africa (1, 2). Indeed, recent studies suggest that hepatitis B virus and AFB1 may exert a synergistic effect (3). Approximately 50%6 of HCC in high AFB1 regions (4, 5), but only 20%o in low AFB1 regions, harbors mutations in the p53 tumor suppressor gene, and the spectrum of mutations is quite different (6, 7). More than half of the tumors from high AFB1 regions contain G --T transversions in the third position of codon 249 (AGG), resulting in the replacement of arginine by serine (4-8). In contrast, mutations are distributed throughout the highly conserved domains IV and V of p53 in HCC from low AFB1 regions and no prevalence of G --T transversions is observed (9, 10). These results indicate that the substitution of arginine 249 by serine in the p53 protein is not required for hepatocarcinogenesis in man. Though G --T transversions are in agreement with the mutational specificity of AFB1 (11, 12) and other carcinogens forming bulky DNA adducts (13,14), there is no convincing explanation for the prevalence of mutations in codon 249, which almost never harbors mutations in other forms of human cancer. Many factors determine the mutability of a particular gene sequence, in addition to the chemical properties of the ultimate mutagen, and it is difficult to predict on the basis of model experiments. Among them, sequence context, local DNA conformation, chromatin structure, transcriptional activity, and repairability are recognized to play a role. Therefore, we have directly evaluated the mutability to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.AFB1 of codons 247-250 in human hepatocarcinoma cells HepG2 by genotypic analysis using Msp I and Hae III restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) (15-17). Our results indicate that AFB1 induces the transversion of G --T in the third position of codon 249, with the highest frequency producing the same mutation that is found almost exclusively in HCC from high AFB1 regions in east Asia and Africa. Our results support the notion that AFB1 represents a causative carcinogen in hepatocarcinogenesis in these regions of the world. MATERIALS AND METHODSHepG2 human hepatocarcinoma cells were grown in minimum essential medium supplemented with 10% fetal calf serum to =60% confluency before treatment for 30 min with AFB1 (0.5 ug/ml) in the presence of rat liver microsomes as described (18)(19)(20). The medium was replaced and cell growth continued for 0 hr or for 96 hr, respectively. Despite AFB1 treatment, the cell number doubled within 96 hr. Control cultures were sham-treated only with rat liver microsomes.

Fifty-eight percent of hepatocellular carcinomas (HCCs) from Qidong, China, contain an AGG to AGT mutation at codon 249 of the p53 tumor suppressor gene, a mutation that is rarely seen in HCCs from Western countries. The population of Qidong is exposed to high levels of aflatoxin B1 (AFB1), a fungal toxin that has been shown to induce the same mutation in cultured human HCC cells. To investigate the role of AFB1 and of these p53 mutations in hepatocarcinogenesis, normal liver samples from the United States, Thailand, and Qidong (where AFB1 exposures are negligible, low and high, respectively) were examined for p53 mutations. The frequency of the AGG to AGT mutation at codon 249 paralleled the level of AFB1 exposure, which supports the hypothesis that this toxin has a causative--and probably early--role in hepatocarcinogenesis.

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.

The EFSA ANS Panel was asked to provide a scientific opinion on the safety of green tea catechins from dietary sources including preparations such as food supplements and infusions. Green tea is produced from the leaves of Camellia sinensis (L.) Kuntze, without fermentation, which prevents the oxidation of polyphenolic components. Most of the polyphenols in green tea are catechins. The Panel considered the possible association between the consumption of (-)-epigallocatechin-3-gallate (EGCG), the most relevant catechin in green tea, and hepatotoxicity. This scientific opinion is based on published scientific literature, including interventional studies, monographs and reports by national and international authorities and data received following a public 'Call for data'. The mean daily intake of EGCG resulting from the consumption of green tea infusions ranges from 90 to 300 mg/day while exposure by high-level consumers is estimated to be up to 866 mg EGCG/day, in the adult population in the EU. Food supplements containing green tea catechins provide a daily dose of EGCG in the range of 5-1,000 mg/day, for adult population. The Panel concluded that catechins from green tea infusion, prepared in a traditional way, and reconstituted drinks with an equivalent composition to traditional green tea infusions, are in general considered to be safe according to the presumption of safety approach provided the intake corresponds to reported intakes in European Member States. However, rare cases of liver injury have been reported after consumption of green tea infusions, most probably due to an idiosyncratic reaction. Based on the available data on the potential adverse effects of green tea catechins on the liver, the Panel concluded that there is evidence from interventional clinical trials that intake of doses equal or above 800 mg EGCG/day taken as a food supplement has been shown to induce a statistically significant increase of serum transaminases in treated subjects compared to control.

The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of silicon dioxide (E 551) when used as a food additive. The forms of synthetic amorphous silica (SAS) used as E 551 include fumed silica and hydrated silica (precipitated silica, silica gel and hydrous silica). The Scientific Committee on Food (SCF) established a group acceptable daily intake (ADI) 'not specified' for silicon dioxide and silicates. SAS materials used in the available biological and toxicological studies were different in their physicochemical properties; their characteristics were not always described in sufficient detail. Silicon dioxide appears to be poorly absorbed. However, silicon-containing material (in some cases presumed to be silicon dioxide) was found in some tissues. Despite the limitations in the subchronic, reproductive and developmental toxicological studies, including studies with nano silicon dioxide, there was no indication of adverse effects. E 551 does not raise a concern with respect to genotoxicity. In the absence of a long-term study with nano silicon dioxide, the Panel could not extrapolate the results from the available chronic study with a material, which does not cover the full-size range of the nanoparticles that could be present in the food additive E 551, to a material complying with the current specifications for E 551. These specifications do not exclude the presence of nanoparticles. The highest exposure estimates were at least one order of magnitude lower than the no observed adverse effect levels (NOAELs) identified (the highest doses tested). The Panel concluded that the EU specifications are insufficient to adequately characterise the food additive E 551. Clear characterisation of particle size distribution is required. Based on the available database, there was no indication for toxicity of E 551 at the reported uses and use levels. Because of the limitations in the available database, the Panel was unable to confirm the current ADI 'not specified'. The Panel recommended some modifications of the EU specifications for E 551.

Following a request from the European Commission, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion re-evaluating the safety of microcrystalline cellulose (E 460(i)), powdered cellulose (E 460(ii)), methyl cellulose (E 461), ethyl cellulose (E 462), hydroxypropyl cellulose (E 463), hydroxypropyl methyl cellulose (E 464), ethyl methyl cellulose (E 465), sodium carboxy methyl cellulose (E 466), enzymatically hydrolysed carboxy methyl cellulose (E 469) and cross-linked carboxy methyl cellulose (E 468) as food additives. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) established an acceptable daily intake (ADI) 'not specified' for unmodified and modified celluloses. Celluloses are not absorbed and are excreted intact in the faeces; in addition, microcrystalline cellulose, powdered and modified celluloses could be fermented by the intestinal flora in animals and humans. Specific toxicity data were not always available for all the celluloses evaluated in the present opinion and for all endpoints. Given their structural, physicochemical and biological similarities, the Panel considered it possible to readacross between all the celluloses. The acute toxicity of celluloses was low and there was no genotoxic concern. Short-term and subchronic dietary toxicity studies performed with E 460(i), E 461, E 462, E 463, E 464, E 466 and E 469 at levels up to 10% did not indicate specific treatment related adverse effects. In chronic toxicity studies performed with E 460(i), E 461, E 463, E 464, E 465 and E 466, the no observed adverse effect level (NOAEL) values reported ranged up to 9,000 mg/kg body weight (bw) per day. No carcinogenic properties were detected for microcrystalline cellulose and modified celluloses. Adverse effects on reproductive performance or developmental effects were not observed with celluloses at doses greater than 1,000 mg/kg bw by gavage (often the highest dose tested). The combined exposure to celluloses (E 460-466, E 468 and E 469) at 95th percentile of the refined (brand-loyal) exposure assessment for the general population was up to 506 mg/kg bw per day. The Panel concluded that there was no need for a numerical ADI and that there would be no safety concern at the reported uses and use levels for the unmodified and modified celluloses (E 460(i); E 460(ii); E 461-466; E 468 and E 469). The Panel considered an indicative total exposure of around 660-900 mg/kg bw per day for microcrystalline, powdered and modified celluloses.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12564 Da and a molar absorption coefficient at 280 nm of 17700 M−1 cm−1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin. A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in λgt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably consitutes the N‐terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5‐kDa protein.