[Chemical food safety : National and European aspects]. / Chemische Lebensmittelsicherheit : Nationale und europäische Aspekte.

Chemical food safety deals with the health evaluation of compounds in food with regard to toxicological aspects. In the following, examples of current interest from various categories of compounds in foods, e.g., of naturally occurring substances and of heat-induced or process-related contaminants, are presented and current problems in their toxicological evaluation are described. To guarantee that human intake of such compounds will occur in safe amounts only, an assessment of their health risks based on the present state of science and according to internationally recognized methods has to be provided. This risk assessment is independent and is performed at the national level by the Federal Institute for Risk Assessment and at the European level by the European Food Safety Authority. Results and findings of the risk assessment of toxicologically relevant compounds are the scientific basis for recommendations and strategies for consumer protection. For example, measures like the setting of maximum levels for contaminants in certain food categories can be the result. At the national level, the Federal Office for Consumer Protection and Food Safety is responsible for risk management, while at the European level the European Commission and other institutions develop the measures for the member states.

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Histological alterations in ovaries of pubertal female rats induced by triphenyltin.

Triphenyltin is an organotin compound that has been used extensively as an antifouling biocide and as an agricultural pesticide. Certain organotin compounds act as endocrine-active agents and have been reported to affect reproduction in mollusks and mammals. Here we studied the histopathological effects of 2 or 6mg triphenyltin chloride (TPTCl)/kg b.w. on the reproductive tissue and the thymus of female pubertal rats as part of a comprehensive pubertal assay. Beginning at postnatal day (PND) 23 female Wistar rats were treated daily per gavage until their first estrus after PND 53. Reproductive organs were removed and histologically evaluated. While no histological changes were observed in oviduct, uterus, vagina and mamma, an increase in the number of all follicle stages occurred at both dose levels. Furthermore, exposure to 2mg TPTCl/kg b.w. led to a significant reduction in the diameter of tertiary follicles. A significant increase in the number of atretic follicles was observed in tertiary and preovulatory follicles after exposure to 6mg TPTCl. The thymus displayed a decreased number of apoptotic cells in both dose groups. We conclude that peripubertal administration of 2 and 6mg TPTCl/kg b.w. caused effects on ovarian follicles of female rats.

Proliferation and differentiation of murine haemopoietic progenitor cells in stroma-free culture in the presence of metabolites of chlorinated pesticides.

We have studied the influence of metabolites of chlorinated pesticides (lindane, pentachlorophenol, hexachlorobenzene) on proliferation and differentiation in two stroma-free murine bone marrow culture models, a multipotent progenitor cell line (FDCP-mix) and primary lineage-depleted bone marrow cells. Tetrachlorohydroquinone (Cl(4)pHQ), tetrachloro-p-benzoquinone (Cl(4p)BQ), but not their positional isomers, tetrachlorocatechol (Cl(4)oHQ) and tetrachloro-o-benzoquinone (Cl(4)oBQ), nor 2,4,6-trichlorophenol (2,4,6-Cl(3)P), were much more toxic to FDCP-mix cells cultured under conditions which lead to self-renewal than under conditions which lead to granulocyte-macrophage differentiation. Under the latter conditions, Cl(4)pHQ and Cl(4p)BQ even stimulated growth at intermediate concentration levels. In the primary cell cultures, pronounced differences were observed in the sensitivity between individual developmental pathways and between the different compounds. The percent of cells differentiating into the granulocytic lineage was increased at high concentration levels of each test compound. However, stimulatory effects on the macrophage lineage were observed at intermediate concentration levels of Cl(4)pHQ, Cl(4p)BQ and 2,4,6-Cl(3)P, and differentiation into erythrocytes was stimulated at low concentrations of 2,4,6-Cl(3)P. It is concluded that chlorinated monocyclic pesticides, after biotransformation to quinoid metabolites, may interact directly with haemopoietic progenitor cells with differential effects on self-renewal and differentiation. These mechanisms could lead to myeloplastic disorders.

The carcinogenicity of the biocide ortho-phenylphenol.

The biocides ortho-phenylphenol and its sodium salt (OPP and SOPP) are widely used as fungicides and antibacterial agents for commercial and consumer purposes. The carcinogenicity of OPP/SOPP toward the urinary bladder was demonstrated when rats were chronically fed concentrations of 0.5%-4% in their diet. Other species tested so far did not develop tumours. Understanding the mechanisms underlying OPP/SOPP-induced bladder carcinogenesis is critical to determine whether risks observed at high doses in rats are of relevance to humans exposed at much lower levels. This overview details experimental studies of carcinogenicity, genotoxicity as well as metabolism/toxicokinetics and other mechanistic studies which bear on cancer hazard and risk evaluation of exposure to humans. Based on the presently available knowledge, it is concluded that reactive quinoid metabolites exhibiting redox cycling activities are the crucial factors. At certain concentration levels, these metabolites are able to produce cytotoxic events with concomitant enhanced cell proliferation of the target tissue. Further important risk factors are probably promutagenic lesions induced by oxidative stress and a higher urinary pH. Supposed that these mechanisms are the basis for the tumourigenicity observed, then suitable low doses of OPP/SOPP will practically pose no cancer risk.

Metabolites of the biocide o-phenylphenol generate oxidative DNA lesions in V 79 cells.

Incubation of the o-phenylphenol (OPP) metabolites, o-phenylhydroquinone (PHQ) and o-phenylbenzoquinone (PBQ) with V 79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in nuclear DNA. With OPP no distinct induction of this lesion could be observed. In addition, PHQ and PBQ were able to generate DNA single-strand breaks (DNA SSB), while OPP failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activations and the lowest effective concentration tested was 20 microM. It is concluded that these metabolites may contribute to the carcinogenicity of OPP and sodium o-phenylphenolate (SOPP) observed in rats, by generating reactive oxygen species (ROS) through their redox cycling properties.

Oxidative DNA lesions in V79 cells mediated by pentachlorophenol metabolites.

Incubation of the pentachlorophenol (PCP) metabolites, tetrachloro-p-benzoquinone (chloranil, TCpBQ), tetrachloro-p-hydroquinone (TCpHQ) and tetrachloro-p-benzoquinone (TCoBQ) with V79 Chinese hamster cells led to a significant enhancement of the amount of 8-hydroxydeoxyguanosine (8-OH-dG) in DNA. With PCP itself and the metabolite tetrachloro-o-hydroquinone (TCoHQ) no distinct induction of this lesion could be observed. The average yields of 8-OH-dG were about 2-2.5 times above background levels. In addition, TCpBQ and TCpHQ were able to generate DNA single-strand breaks, while PCP, TCoHQ and TCoBQ failed to induce this lesion. All incubations were performed for 1 h without exogenous metabolic activation and concentrations were 25 microM of the respective agent. It is concluded that these metabolites may contribute to the carcinogenicity of PCP observed in mice, by generating reactive oxygen species (ROS) through their redox cycling properties.

Induction of 8-hydroxy-2-deoxyguanosine and single-strand breaks in DNA of V79 cells by tetrachloro-p-hydroquinone.

The biocide pentachlorophenol (PCP) is in part metabolized, by microsomal enzymes, to tetrachloro-p-hydroquinone (TCHQ), which easily oxidizes to its semiquinone radical. Redox cycling of this compound produces reactive oxygen species (ROS) which ultimately may damage cellular DNA. Here, we report on DNA damage generated by TCHQ in hamster lung fibroblasts (V79 cells) using 8-hydroxy-2-deoxyguanosine (8-OH-dG) as a marker of a major oxidative genetic lesion and measuring the induction of DNA single-strand breaks (DNA SSB) with the aid of the alkaline elution assay. TCHQ was administered to cell cultures in concentrations of 6.25, 12.5, 25, and 50 microM for 1 h. 6.25 and 12.5 microM had no significant effect on both parameters, whereas the higher concentrations resulted in increases of the level of 8-OH-dG (2.3- and 2.0-fold, respectively) and induced DNA SSB. The latter lesion may arise from (i) direct attack of OH., (ii) repair of hydroxylated DNA bases, or (iii) cytotoxic effects. Metabolic transformation of PCP to TCHQ and/or other metabolites with quinoid structures and the subsequent generation of ROS, producing oxidative DNA damage, may play a role in PCP-induced carcinogenicity observed in mice.

The pentachlorophenol metabolite tetrachloro-p-hydroquinone induces the formation of 8-hydroxy-2-deoxyguanosine in liver DNA of male B6C3F1 mice.

Tetrachloro-p-hydroquinone (TCHQ), the major metabolite of pentachlorophenol (PCP) in mammalian systems, is known to autoxidize to its semiquinone radical under physiological conditions. In this way, PCP could present a potent source of reactive oxygen species (ROS) during metabolization. ROS contribute to numerous modifications of DNA. Formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a product of hydroxyl radical attack on DNA, is monitored as a marker of a major genetic lesion induced by agents which produce oxygen radicals. We studied the properties of TCHQ for the induction of oxidative DNA damage in vivo. Male B6C3F1 mice were fed a diet containing TCHQ for 2 and 4 weeks. These experiments resulted in an enhancement of about 50% of the 8-OH-dG portion in liver DNA after administration of 300 mg TCHQ/kg body wt./day for 2 weeks. Control levels did not change over the periods of 2 and 4 weeks, respectively. In contrast to these results, a single i.p. injection of 20 or 50 mg/kg body wt. did not affect the 8-OH-dG content after 6 and 24 h, respectively. These data may support a possible contribution of ROS to the carcinogenicity of PCP.

NO2-induced DNA single strand breaks are inhibited by antioxidative vitamins in V79 cells.

Recently, we were able to show that nitrogen dioxide (NO2), a strong oxidant, induced DNA single strand breaks (SSBs) in V79 cells. Possibly, special scavengers, e.g. antioxidative vitamins, may protect cells from NO2-induced damage. Therefore, the effect of various tocopherols, beta-carotene, retinol, and ascorbic acid on NO2-induced SSBs in V79 cells was investigated. Cells were preincubated with vitamins and treated for 10 min with 200 ppm NO2. The rate of SSBs was measured by the alkaline elution assay, the amount of DNA by a fluorimetric assay. Micromolar concentrations of d-gamma-tocopherol inhibited the rate of NO2-induced SSBs by 40%, beta-carotene and ascorbic acid by 25%. None of these vitamins had any effects on DNA or the viability of cells. When incubating the cells with retinol in a medium with pH 8.5, this vitamin inhibited NO2-induced SSBs, reducing them by 35%. However, in high concentrations, retinol itself induced SSBs and influenced cell viability. The results are discussed with regard to many toxic effects of NO2.

N-nitrosodimethylamine, N-nitrosodiethylamine, and N-nitrosomorpholine fail to generate 8-hydroxy-2'-deoxyguanosine in liver DNA of male F344 rats.

The possible oxidative effects of the hepatocarcinogens 2-nitropropane (2-NP), N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosomorpholine (NMOR) on nuclear DNA were studied in vivo in male F344 rats. 2-NP and the N-nitrosamines were administered intraperitoneally. In addition, NDEA was given by gavage. DNA was isolated from rat liver and hydrolysed enzymatically. Oxidative DNA damage was determined by measuring 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in a mixture of 2'-deoxyribonucleosides by electrochemical detection. This method allows a detection limit of about 0.1 residue 8-OH-dG per 10(5) 2'-deoxyguanosine (dG). 2-NP drastically increased the content of 8-OH-dG in rat liver by a factor of ca. 12. No elevation above control values could be proved after having dosed the rats with N-nitrosamines.

Effect of varying the concentration of phenobarbital and its duration of treatment on the evolution of carcinogen induced enzyme-altered foci in rat liver.

The present study was aimed to investigate whether the promoting activity of phenobarbital in rodent liver is related to its daily dose level and duration of treatment or rather to its total dose administered. For this purpose groups of female Wistar rats were treated for 5 consecutive days with an initiating dose of 10 mg/kg body weight N-nitrosodiethylamine. Subsequently, rats were given phenobarbital-sodium (PB) in their drinking water at concentrations of 20, 50, 100 and 200 mg/l for varying lengths of time, such that the total dose of xenobiotic was very similar throughout the different treatment groups ranging from approximately 950 to 1100 mg/kg body weight. The number and volume fraction of lesions negative for the marker enzyme adenosine triphosphatase in liver were subsequently scored as a means to determine the carcinogenic response in this organ. Slight promoting effects of PB were only seen at the lowest concentration of 20 mg/l, whereas no significant effects were observed at 50 and 100 mg/l. At the highest concentration of 200 mg/l an inhibition of carcinogenic response was obtained. Although the effects seen in this study were only moderate, our data favour the idea that the promoting effects of PB depend on the actual concentration of the compound and the duration of treatment rather than on the total dose administered.

Metabolic denitrosation of N-nitroso-N-methylaniline: detection of amine-metabolites.

The enzymatic denitrosation of N-nitroso-N-methylaniline (NMA) was investigated by measuring the resulting amine metabolites when NMA was incubated with liver microsomes of PB-pretreated mice. Aniline was found to be the main amine metabolite. Small amounts of the secondary amine, N-methylaniline (MA) and its metabolite, p-methylaminophenol (p-MAP), could also be detected. Incubation of MA resulted in the formation of aniline and p-MAP. The velocity of the metabolism of MA was somewhat faster than that of NMA. On the basis of the measured Vmax values the formation of aniline from MA or from NMA proceeded at nearly identical rates. The dissociation constants as a measure of binding affinity to cytochrome (cyt.) P-450 were determined by measuring the binding spectra. NMA has one Ks of 3.1 mM, whereas MA shows two apparent Ks values, 650 microM and 25 mM, respectively. The results are discussed in relation to the enzymatic mechanism of denitrosation of NMA.

Metabolic denitrosation of N-nitrosamines: mechanism and biological consequences.

NADPH-dependent microsomal metabolism of N-nitrosamines results in both oxidative dealkylation and denitrosation of the molecule. For denitrosation, two enzymatic mechanisms have been proposed: (i) cytochrome P450 (P450)-dependent one-electron reduction of the nitrosamine molecule, resulting in the formation of nitric oxide (NO) and secondary and primary amine, and (ii) liberation of NO via an oxidative mechanism mediated by a P450-dependent one-electron abstraction. In order to clarify the mechanism of denitrosation, the metabolism and kinetics of N-nitrosodibenzylamine (NDBzA) and its corresponding secondary amine dibenzylamine were studied. The main metabolites of NDBzA are benzaldehyde, the primary amine benzylamine and nitrite. An important finding is that benzaldehyde is generated more rapidly from dibenzylamine than from the parent NDBzA. During reductive denitrosation of NDBzA, the oxygen atom in benzaldehyde is derived from air, while benzaldehyde generated via the oxidative mechanism of denitrosation receives its oxygen atom from water due to hydrolysis of the intermediary benzylidenebenzylamine. Microsomal incubation of NDBzA in buffer containing 18O-H2O resulted in no incorporation of 18O from water into benzaldehyde, which could be related to the formation of the corresponding benzaldehyde, which could be related to the formation of the corresponding benzylidenebenzylamine. It is concluded that NDBzA is denitrosated by the proposed reductive mechanism. Current belief is that denitrosation leads to detoxification of the NA molecule; however, toxic effects cannot be excluded if the conversion of NO into NO2- and NO3- involves intermediary formation of the NO2 radical.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA damage induced by N-nitrosodibenzylamine and N-nitroso-alpha-acetoxybenzyl-benzylamine in mammalian cell systems and in vivo.

N-nitrosodibenzylamine (NDBzA) and N-nitroso-alpha-acetoxybenzyl-benzylamine (alpha-acetoxy-NDBzA) were tested for induction of DNA single-strand breaks (SSBs) in V 79 Chinese hamster cells (V 79 cells) and isolated rat hepatocytes. The alkaline elution assay was used for the detection of DNA strand breaks. Treatment of V 79 cells with alpha-acetoxy-NDBzA effectively increased the rate of DNA SSBs, while with NDBzA, no DNA-damaging activity was detectable. Both substances produced significant DNA damage in rat hepatocytes. Interestingly, NDBzA was able to induce SSBs at significantly lower concentrations than alpha-acetoxy-NDBzA. The possible reasons for these findings are discussed. In contrast to these in vitro results, NDBzA exhibited very weak in vivo activity.

Nitrogen dioxide induces DNA single-strand breaks in cultured Chinese hamster cells.

Gaseous nitric oxide (NO) and nitrogen dioxide (NO2) were tested for their potential to induce DNA single-strand breaks (SSBs) in Chinese hamster cells (V79 cells). The alkaline elution assay was used for the detection of SSBs. V79 cells were exposed to NO and NO2 in N2 in varying concentrations (0-500 p.p.m.) and over varying periods (5-30 min). NO treatment did not result in any detectable DNA damage. NO2 led to a dose- and time-dependent increase of the rate of SSBs and the amount was dependent on concentration of NO2 and the length of exposure. The lowest observable effective concentration which was statistically different from control values was 10 p.p.m. exposed for 20 min. The metabolites of both gases, nitrite (NO2-) and nitrate (NO3-), had no DNA-damaging activity up to a concentration of 1 mM. The mechanism by which NO2 may generate SSBs is discussed.

Enhanced demethylation and denitrosation of N-nitrosodimethylamine by human liver microsomes from alcoholics.

The metabolism of N-nitrosodimethylamine (NDMA) was investigated in incubations with human liver microsomes from alcoholics and control patients who suffered from other diseases, but had a histological normal liver. All of the microsomal samples studied were able to metabolize NDMA at various concentrations to both formaldehyde and nitrite. Analysis of the liver microsomes from alcoholics revealed that both enzymatic activities--formaldehyde and nitrite formation--were enhanced several times as compared to the control patients. The results point to the fact that alcoholics metabolize NDMA at a higher rate probably due to the induction of one or more ethanol-inducible human liver cytochromes (cyt.) P450. The question if alcoholics therefore possess a higher risk for carcinogenic events is discussed.