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In this review, current issues and opportunities in food safety assessment are discussed. Food safety is considered an essential element inherent in global food security. Hazard characterization is pivotal within the continuum of risk assessment, but it may be conceived only within a very limited frame as a true alternative to risk assessment. Elucidation of the mode of action underlying a given hazard is vital to create a plausible basis for human toxicology evaluation. Risk assessment, to convey meaningful risk communication, must be based on appropriate and reliable consideration of both exposure and mode of action. New perspectives, provided by monitoring human exogenous and endogenous exposure biomarkers, are considered of great promise to support classical risk extrapolation from animal toxicology.

Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to 85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals.

In vitro methods are common and widely used for screening and ranking chemicals, and have also been taken into account sporadically for risk assessment purposes in the case of food additives. However, the range of food-associated compounds amenable to in vitro toxicology is considered much broader, comprising not only natural ingredients, including those from food preparation, but also compounds formed endogenously after exposure, permissible/authorised chemicals including additives, residues, supplements, chemicals from processing and packaging and contaminants. A major promise of in vitro systems is to obtain mechanism-derived information that is considered pivotal for adequate risk assessment. This paper critically reviews the entire process of risk assessment by in vitro toxicology, encompassing ongoing and future developments, with major emphasis on cytotoxicity, cellular responses, toxicokinetics, modelling, metabolism, cancer-related endpoints, developmental toxicity, prediction of allergenicity, and finally, development and application of biomarkers. It describes in depth the use of in vitro methods in strategies for characterising and predicting hazards to the human. Major weaknesses and strengths of these assay systems are addressed, together with some key issues concerning major research priorities to improve hazard identification and characterisation of food-associated chemicals.

The Senate Commission on Food Safety (SKLM, Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln) of the German Research Foundation (DFG, Deutschen Forschungsgemeinschaft) is a transdisciplinary expert committee, providing advice on food safety to the government, parliament, and authorities. Consultation is based on a scientific assessment with the aim to give expert advice to authorities, so that they can make appropriate decisions. The SKLM is independent in its scientific deliberations and under no directive in the selection of issues to work on. Topics considered may result from requests of the Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV, Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz). Other issues may be raised by the SKLM, if they are regarded to be of particular importance for consumer health protection. Issues encompass the scientific assessment of safety and nutritional benefit of food ingredients and additives, of novel and functional food, as well as of novel food technologies. The SKLM identifies gaps in knowledge, research needs, and need for action.

Acrylamide (AA) is a carcinogen as demonstrated in animal experiments, but the relevance for the human situation is still unclear. AA and its metabolite glycidamide (GA) react with nucleophilic regions in biomolecules. However, whereas AA and GA react with proteins, DNA adducts are exclusively formed by GA under conditions simulating in vivo situations. For risk assessment it is of particular interest to elucidate whether AA or GA within the plasma concentration range resulting from food intake are "quenched" by preferential reaction with non-critical blood constituents or whether DNA in lymphocytes is damaged concomitantly under such conditions. To address this question dose- and time-dependent induction of hemoglobin (Hb) adducts as well as genotoxic and mutagenic effects by AA or GA were studied in human blood as a model system.

The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) advises authorities and the government on food safety. The risk assessment of foodstuffs, including novel and functional foods, covers the evaluation of food ingredients and additives as well as the evaluation of novel processing methods. In carrying out this task the Commission expresses its opinion primarily on those aspects which are concerned with the safety assessment. In addition, questions relating to the technological need and to nutritional or physiological benefits are also considered. The topics considered may originate from enquiries of the Ministry for Consumer Protection, Nutrition and Agriculture (BMVEL). Other topics may be selected by the Commission on its own initiative, if they are considered to be of particular importance for consumer protection. Within the context of this activity the SKLM also organises symposia and expert discussions, their outcome subsequently being published as resolutions, conclusions or opinions.

Die Senatskommission zur Beurteilung der gesundheitlichen Unbedenklichkeit von Lebensmitteln (SKLM) der Deutschen Forschungsgemeinschaft (DFG) berat Behorden und Regierung in Fragen der gesundheitlichen Unbedenklichkeit von Lebensmitteln. Die gesundheitliche Bewertung von Lebensmitteln, unter Einschluss neuartiger und funktioneller Lebensmittel, betrifft die Beurteilung von Inhalts- und Zusatzstoffen, aber auch die Bewertung neuartiger Lebensmitteltechnologien/Verfahren. Dabei ausert sich die Kommission primar zu Aspekten der Sicherheitsbewertung, berucksichtigt jedoch auch Fragen der technologischen Notwendigkeit und des ernahrungsphysiologischen Nutzens. Konkrete Themen ergeben sich aus aktuellen Anfragen des Bundesministeriums fur Verbraucherschutz, Ernahrung und Landwirtschaft (BMVEL). Sie werden aber auch von der Kommission selbst aufgegriffen, wenn ihnen besondere Bedeutung fur den Verbraucherschutz zugemessen wird. Im Rahmen dieser Tatigkeit organisiert die SKLM auserdem Symposien und Expertengesprache, deren Ergebnisse in Form von Beschlussen, Stellungnahmen und Mitteilungen veroffentlicht werden.

Alpha,beta-unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 microM) and CHX (300 microM), HEXDI and CA (300 microM each), tGSH was depleted down to20% of control (viability85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals.

This report presents a review of risk characterisation, the final step in risk assessment of exposures to food chemicals. The report is the second publication of the project Food Safety in Europe: Risk Assessment of Chemicals in the Food and Diet (FOSIE). The science underpinning the hazard identification, hazard characterisation and exposure assessment steps has been published in a previous report (Food Safety in Europe, 2002). Risk characterisation is the stage of risk assessment that integrates information from exposure assessment and hazard characterisation into advice suitable for use in decision-making. The focus of this review is primarily on risk characterisation of low molecular weight chemicals, but consideration is also given to micronutrients and nutritional supplements, macronutrients and whole foods. Problem formulation, as discussed here, is a preliminary step in risk assessment that considers whether an assessment is needed, who should be involved in the process and the further risk management, and how the information will provide the necessary support for risk management. In this step an evaluation is made of whether data are available and what level of resources are needed, as well as the timeline for completing the assessment. The report describes good evaluation practice as an organisational process and the necessary condition under which risk assessment of chemicals should be planned, performed, scrutinised and reported. The outcome of risk characterisation may be quantitative estimates of risks, if any, associated with different levels of exposure, or advice on particular levels of exposure that would be without appreciable risk to health, e.g. a guidance value such as an acceptable daily intake (ADI). It should be recognised that risk characterisation often is an iterative and evolving process. Historically, different approaches have been adopted for the risk characterisation of threshold and non-threshold effects. The hazard characterisation for threshold effects involves the derivation of a level of exposure at or below which there would be no appreciable risk to health if the chemical were to be consumed daily throughout life. A guidance value such as the ADI, is derived from the no-observed-adverse-effect-level (NOAEL) or other starting point, such as the benchmark dose (BMD), by the use of an uncertainty or adjustment factor. In contrast, for non-threshold effects a quantitative hazard estimate can be calculated by extrapolation, usually in a linear fashion, from an observed incidence within the experimental dose-response range to a given low incidence at a low dose. This traditional approach is based on the assumption that there may not be a threshold dose for effects involving genotoxicity. Alternatively, for compounds that are genotoxic, advice may be given that the exposure should be reduced to as low as reasonably achievable (ALARA) or practicable (ALARP). When a NOAEL can be derived from a study in humans, this would be utilised in the derivation of guidance values or advice. However, there may be uncertainties related to the possible role of confounders and the precision of both the incidence and exposure data. Individuals may be at an increased risk because of their greater exposure or their greater sensitivity. Risk characterisation should include information not only on the general population, but also on any subpopulation considered to be potentially susceptible.

Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2.Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten.The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.

E91 (17 beta-[N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-alanyl]-5 alpha-dihydrotestosterone) (CNC-ala-DHT) is a newly synthesised alkylating compound consisting of N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-alanine (CNC-ala) as the alkylating moiety and of 5 alpha-dihydrotestosterone (DHT) as a steroid carrier molecule. We studied the antitumour activity of E91 (final concentrations: 0.1, 1, 10 and 30 mumol/l) against freshly explanted human tumours, using an in vitro soft agar cloning system. A total of 54 tumour samples was evaluated using 1 h-exposure and 51 tumour specimens were studied using a continuous exposure for 21-28 days. In addition, the compound's activity was compared with other clinically used anticancer agents. After short-term exposure, 49 of 53 evaluable specimens (92%) had adequate colony formation, as compared with 49 of 50 (98%) after long-term exposure. After short-term exposure, E91 exhibited only marginal antitumour activity. However, in long-term exposure experiments, E91 had marked and concentration-dependent antitumour activity (P0.001). At concentrations of10 mumol/l, E91 was as active as the other clinically used antineoplastic agents and at 30 mumol/l, E91 was significantly more active than 5-fluorouracil (P = 0.041). E91 showed activity against a wide spectrum of tumour types. The highest activity was observed against colorectal carcinomas (3/4 tumour specimens inhibited at 30 mumol/l). Sensitivity was also high remarkable in breast cancer specimens with 3/6 specimens inhibited at 30 mumol/l. In vitro myelotoxicity was less than that of doxorubicin. At 30 mumol/l, E91 induced a reduction of colony forming units-granulocyte macrophage (CFU-GM) to only 53% of control and of CFU-GEMM to 20% of control. We conclude that because of broad activity and reduced myelotoxicity further clinical development of E91 appears warranted.

The novel steroid conjugates 17 beta-[N-[N'-(2-chloroethyl)-N'-nitroso] carbamoyl]-glycyl-19-nortestosterone (1) and 17 beta-[N-[N'-(2-chloroethyl)-N'-nitroso]carbamoyl]-L-alyanyl-19- nortestosterone (2) were synthesized and characterized with respect to affinity for steroid receptors and for androgenic efficacy. At an i.p. dosage of 50 mg/kg, conjugates 1 and 2 induced strong tumor inhibition of Nobel Nb prostate carcinoma in rats, but also a marked loss of body weight. In two further experiments, treatment with conjugate 2 at a dosage of 25 mg/kg demonstrated high antitumor activity without indication of toxicity. Conjugate 2 achieved the same tumor growth inhibition as a nearly twofold molar dose of cyclophosphamide. The results indicate reproducibly high antitumor activity of 2 in the Noble Nb model at a well tolerated dosage. A low dose equimolar mixture of unlinked N-(2-chloroethyl)-N-nitrosocarbamoyl (CNC)-alanine and 19-nortestosterone was significantly more toxic than conjugate 2, showing about the same adverse effect on the body weight as the conjugate at high dosage. CNC-L-alanine at equimolar dosage was highly toxic, causing early death of all animals.

Maligne Erkrankungen stellen derzeit nach Herz-Kreislauf-Erkrankungen die zweithaufigste Todesursache in den Industrielandern dar und machen etwa 20%–25% aller Todesfalle aus. Etwa 1 Million neue Krebserkrankungen werden jahrlich in den USA diagnostiziert, die Anzahl krebsbedingter Todesfalle pro Jahr betragt etwa eine halbe Million [66]. Die Tumor- inzidenz bzw. -mortalitat in den EU-Landern ist ahnlich: In den 12 Landern der Europaischen Union mit einer gesamten Population von etwa 327 x 106 werden jahrlich 1,2 Millionen Neuerkrankungen sowie etwa 800.000 krebsbedingte Todesfalle gezahlt [8, 34, 36].

In a laboratory construction for heterotrophic biological denitrification of drinking water treatment, the formation of nitrite, the potency of nitrosation and the genotoxic activity were tested. Parameter as nitrate concentration, the water flow rate in the system, nitrite and morpholine addition and the pH-value were checked. For testing the potency of nitrosation and formation of nitrite in the reactor we took morpholine, a fast nitrosing amine. The results show for the break down rate of nitrate, there is no influence of the initial nitrate concentration (80-195 mg/L), the nitrite addition (5-20 mg/L) and the water flow rate (45-100 min) in the system. pH-values below 5.5 showed a little break down rate of nitrate. There was no correlation between the starting point of nitrate concentration and the formation of nitrite, although there was a positive correlation between the length of stay and the formation of nitrite. Nitrite concentrations of 5 mg/L with morpholine concentrations of 5 and 10 mg/L didn't show detectable formation of nitrosomorpholine. The analyses of different watertests in the construction didn't show significant results for DNA damage by the sister-chromatid exchange (SCE). The results of the Salmonella microsome assay (tester strain TA1535) didn't show any mutagenic effects relating to the potency of nitrosation. According to our experiments the potency of generalising nitrosamides or nitrosamines by drinking water denitrification seems to be low. There is no final assessment of detriment to health by denitrifying drinking water.

Isoenzymes of 3',5'-cyclic nucleotide phosphodiesterase (PDE) have been characterized in B16 murine melanoma cells and MCF-7 human mammary carcinoma cells. Separation of soluble phosphodiesterase activity by fast protein liquid chromatography on a Mono-Q column resolved three isoenzymes, MCF-7 cells contained a cyclic GMP-specific isoenzyme (PDE-V), a cyclic GMP-activable isoenzyme (PDE-II), and a cyclic AMP-specific isoenzyme (PDE-IV). B16 cells contained a cyclic GMP-specific isoenzyme (PDE-V), a Ca2+/calmodulin-activated isoenzyme (PDE-I), and a cyclic AMP-specific isoenzyme (PDE-IV). A series of PDE inhibitors was tested for their activity spectrum on PDE isoenzymes. Inhibition of PDE activity in B16 cells by the new compound DC-TA-46, was found to result specifically from PDE-IV inhibition [50% inhibition (IC50) = 0.03 microM]. Much lower inhibitory activity was observed for DC-TA-46 toward PDE-I (IC50 = 5 microM) and PDE-V (IC50 = 14 microM). DC-TA-46 was found to inhibit growth of B16 melanoma and MCF-7 mammary carcinoma cells dose dependently (B16: IC50 = 1.7 microM, MCF-7: IC50 = 2 microM). At 2 microM concentration, growth inhibition of B16 melanoma cells was 60%, concomitant with a decrease in PDE activity of 63% and an increase in cAMP level of 59%. In contrast, incubation with inhibitors specific for PDE-I and PDE-V resulted only in marginal or undetectable growth inhibition. The results suggest a correlation between PDE-IV inhibition and growth inhibition. PDE-IV thus appears to be a potential new target for antiproliferative treatment.

The in vitro chemosensitivity of MAC 15A ascites cells to CNC-alanylalanine and CNC-glycinemethylamide was assessed using a clonogenic assay system. In vitro stability studies and in vivo pharmacokinetics were performed using a reversed-phase HPLC technique. Initial concentrations of CNC-alanylalanine and CNC-glycinemethylamide of 5.2 micrograms ml-1 and 3.2 micrograms ml-1 respectively, were required for a 70% reduction in colony formation of MAC 15A cells in vitro. The concentrations of active alkylating species generated were calculated from the drug half-lives in tissue culture medium. On this basis, a 70% cell kill was achieved by equivalent concentrations of 10.8 microM CNC-alanylalanine and 10.6 microM CNC-glycinemethylamide. Analysis of drug levels following intraperitoneal administration revealed that CNC-alanylalanine was cleared more slowly from the peritoneal cavity producing a greater drug concentration at the site of the ascitic MAC 15A tumour. These results suggested that the superior activity of CNC-alanylalanine over CNC-glycinemethylamide against MAC 15A in vivo could be attributed mainly to differences in the pharmacokinetic behaviour of the two drugs following intraperitoneal administration and that CNC-alanylalanine might have a role in the treatment of local peritoneal disease.

A method for the determination of long-chain N-nitroso-N-methylalkylamines in a range of commercial cosmetic and cleaning emulsions is described. The clean-up consists of solid-phase extraction and column chromatography. N-Nitroso-N-methyldodecylamine, N-nitroso-N-methyltetradecylamine and N-nitroso-N-methylhexadecylamine are detected by gas-liquid chromatography using a Thermal Energy Analyzer. N-Nitroso-N-ethyldodecylamine is used as an internal standard. Analysis of 86 products produced and marketed in the FRG did not indicate significant contamination. In contrast, of 26 consumer products from the USA, three (one hair-care product and two dishwashing liquids), which contained fatty amine oxides, were contaminated with substantial amounts of N-nitroso-N-ethylalkylamines.

The influence of two CNUs with similar alkylating but strongly different carbamoylating activity towards the glutathione system was investigated in different organs. Both CNUs influence the glutathione system of the bone marrow in a similar manner, irrespective of their carbamoylating potential. In contrast, glutathione reductase activity in the other organs was strongly decreased by the potent carbamoylator BCNU, whereas no or only minor effects were produced by its weakly carbamoylating counterpart HECNU. The results confirm that bone marrow toxicity of CNUs primarily results from alkylation and not from carbamoylation. Other organ-related toxic effects, however, are probably a result of carbamoylating reactions exerted by BCNU. This applies especially to lung toxicity that has been observed frequently as a major side effect in clinical trials with BCNU.

The potent carcinogen N-nitrosodiethanolamine (NDELA) becomes mutagenic to Salmonella typhimurium TA98 and TA100 when activated by alcohol dehydrogenase from yeast or horse liver. Metabolic pathways different from alpha-oxidation might therefore be important for the activation of N-nitroso-2-hydroxyalkylamines such as NDELA. In an in-vitro test system (Namalva cells), neither NDELA nor N-nitrosoethyl-2-hydroxyethylamine was genotoxic, whereas the corresponding metabolites from alcohol dehydrogenase-mediated oxidation, N-nitroso-2-hydroxymorpholine and N-nitrosoethylethanalamine, induced single-strand breaks even at low doses. An immuno-slot-blot assay was used to study the formation of O6-2-hydroxyethyldeoxyguanosine in rat liver after oral administration of different N-nitroso-2-hydroxyalkylamines. When given at equimolar doses (0.375 mmol/kg), DNA hydroxyethylation was considerably lower (6.7 mumol/mol deoxyguanosine) with NDELA than with N-nitrosoethyl-2-hydroxyethylamine (48.7 mumol/mol deoxyguanosine) or N-nitrosomethyl-2-hydroxyethylamine (72.1 mumol/mol deoxyguanosine). N-Nitroso-2-hydroxymorpholine did not form detectable levels of O6-2-hydroxyethyldeoxyguanosine.

The carcinogenic action of N-nitrosodibutylamine in the urinary bladder is related to omega-oxidation of a butyl chain. N-Nitrosobutyl-4-hydroxybutylamine and its proximate metabolite N-nitrosobutyl-3-carboxypropylamine (NBCPA) selectively induce urinary bladder tumours in different animal species. The mechanism by which NBCPA exert its carcinogenic action is not known. We found a small but significant dealkylation of NBCPA with microsomes from rat liver or pig urinary bladder, which could be inhibited by SKF 525A. NBCPA was not mutagenic to Salmonella typhimurium (with or without external metabolizing systems from rat liver or pig urinary bladder) and did not induce DNA strand breaks in tumour cell lines (with or without external activation) or primary cells (rat hepatocytes, pig urinary bladder epithelia). Significant induction of sister chromatid exchange and micronuclei, however, was observed in human tumour cells. N-Nitrosoureas that generate the same electrophiles as NBCPA after alpha- or via beta-oxidation (N-butyl-N-nitrosourea, N-3-carboxypropyl-N-nitrosourea and N-2-oxopropyl-N-nitrosourea) induced single-strand breaks in Namalva cells, the oxopropyl compound being more potent than the butyl or carboxypropyl compounds. Our data suggest that NBCPA is activated via alpha-oxidation in the urinary bladder, even though the activation rate in vitro is so low that a positive response is not detectable by classical short-term tests. Provided that beta-oxidation to a highly genotoxic agent proceeds at an adequate rate, it might also be a relevant activation pathway.

A method has been developed for the determination of the N-nitrosoalkanolamines, N-nitrosodiethanolamine (NDELA) and N-nitrosobis(2-hydroxypropyl)amine (NDHPA) in cosmetics. In model systems, we studied nitrosation of the most relevant precursors by NaNO2, by the preservatives Bronopol and Bronidox and by nitric oxides. Secondary amines were most rapidly nitrosated, and Bronopol, Bronidox and atmospheric nitric oxides appeared to be the most relevant nitrosating agents. In order to remove the most important sources of contamination, the Federal Health Office issued an official recommendation to producers to avoid use of secondary amines in cosmetics (March, 1987). Analysis of cosmetics taken from the German market 6-18 months later showed that only 19/126 samples were contaminated with NDELA (12-235 micrograms/kg or NDHPA (40-215 micrograms/kg). The results reflect a strong downward trend in contamination.

A wide spectrum of human and animal tumour cell lines exhibit significantly lower cAMP levels than normal cells, as a consequence of strongly enhanced activities of a cAMP-specific phosphodiesterase isoenzyme (PDE). Inhibition of this isoenzyme by selective inhibitors results in a long lasting, concentration-dependent rise of intracellular cAMP, accompanied by marked growth inhibition. At higher concentrations of the inhibitor (>3 μM), induction of apoptosis becomes apparent, as detected by flow cytometry, confocal microscopy and ELISA-based determination of fragmented DNA in intact cells. Thus tumour-associated overexpression of a cAMP-specific PDE-isoenzyme in proliferating tumours offers a novel cellular target for selective antineoplastic therapy.

Three water soluble and 6 bifunctional newly synthesized analogs of 1,3 -bis( 2 -chloroethyl)- 1 -nitrosourea (BCNU) were screened for anticancer activity against subcutaneously and intracerebrally inoculated Walker carcinosarcoma 256 and 7,12 -dimethylbenz(a)anthracene (DMBA) induced mammary cancer of the rat. The water soluble 1-(2 -hydroxyethyl)- 3-(2 -chloroethyl)- 3 -nitrosourea showed a higher activity than BCNU in s.c. inoculated Walker carcinosarcoma 256 in several experiments. Treatment of i.c. inoculated Walker carcinosarcoma 256 with the bifunctional analogs and 1-(2 -hydroxyethyl)- 3-(2 -chloroethyl)- 3 -nitrosourea resulted in a marked increase in lifespan over untreated rats. However, statistically no differences were found in comparison to the treatment with BCNU. Against the DMBA induced mammary cancer of the rat, only 2 new analogs were examined; they produced a smaller tumor weight inhibition than BCNU. Further investigations are under work to examine the activity of the new compounds against chemically induced brain tumors.

Three new nitrosoureas and chlorozotocin were screened for anticancer activity against Walker carcinoma 256 and DMBA-induced mammary cancer of the rat. High single doses (80% of LD50) of water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylencarboxamido)urea and 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethansulfonate effected a similar tumor weight inhibition than BCNU in treatment of subcutaneously implanted Walker 256. In dose-response studies low doses of the new analogs effected a higher tumor weight inhibition than BCNU in the treatment of subcutaneously implanted Walker 256. The therapeutic index calculated as LD50/ED50, was 2.7 and 2.4 for the new compounds in comparison to BCNU with 2.1. Chlorozotocin and 1-(methylenecarboxyethyl)-1-nitroso-3-phenylurea were not active. Against DMBA-induced mammary cancer the new BCNU analogs yielded a greater tumor weight inhibition than adriamycin and BCNU. At a dose-level of approximately LD10, the remission rates of individual tumors, which were at least 0.6 g at the beginning of treatment were 46% (13/28) for therapy with 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethanesulfonate, 32% (9/28) for 1-(2-chloroethyl)-1-nitro-3-(methylenecarboxamido)urea, 26% (6/23) for chlorozotocin, 21% (7/33) for adriamycin and 17% for BCNU, respectively. As single agents the 2 new analogs 2[3-(1-chloroethyl)-3-nitrossureido[ethylmethanesulfonate, the water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)urea and chlorozotocin were the most effective compounds against DMBA-induced mammary cancer of the rat.

The pyrolysis of nitrosamines in a modified Hall microelectrolytic conductivity system provides the basis for a highly selective and sensitive determination method. Under the conditions specified, the response of this detector system to nitrosamines is at least 10 7 times greater than that for n -hexane. The influence of several operational parameters on the response of the detector to nitrosamines was studied. For open-chain nitrosamines, the detection limit is 50 pg and the response is linear up to at least 200 ng.

Vegetables and vegetable juices with concentrations of nitrate between 27 and 4031 ppm were ingested by 11 volunteers in 30 experiments. Variations of nitrate and nitrite concentrations in saliva were determined for up to 7 hr at intervals of 30 or 60 min. The increase in the amount of nitrate secreted by the salivary glands was found to depend directly on the amount of nitrate ingested. Nitrite concentration in saliva correlated directly with salivary nitrate content. This suggests that there is a direct relationship between the salivary nitrite concentrations and the amounts of nitrate ingested in the diet. The average increase of nitrite concentration in saliva was 20 ppm/100 mg nitrate ingested. The possible relevance of these findings to the endogenous formation of N-nitroso compounds in the gastro-intestinal tract is discussed.

Mechanisms of DNA adduct formation by antineoplastic 2-chloroethyl-N-nitrosoureas (CNUs) and of DNA damage induced by these compounds are discussed. CNUs are alkylating agents that form DNA-DNA cross-links as well as 2-chloroethylated and 2-hydroxyethylated adducts, the N-7-position of guanine being the predominantly alkylated site. A close correlation exists between the potential of a given compound to induce DNA-DNA cross-links and its antineoplastic effectiveness. However, levels of DNA-DNA cross-linking in bone marrow and extent of myelosuppression as measured in rodents are also closely correlated. The design of new cross-linking analogues capable of directing the antineoplastically relevant activity predominantly to the target tumour appears therefore to be of great promise. Cross-linking agents have been attached to a variety of steroid hormone carrier molecules and the conjugates have been tested in structure-activity studies using hormone-receptor containing animal tumours. These studies have revealed that some hormone-linked antineoplastic agents are highly effective in receptor positive experimental tumours and are superior to mixtures of unlinked alkylating agents with hormones. Indications for a relative enrichment of DNA damaging effects in the tumour tissue and for reduced myelotoxicity have been obtained with specific hormone conjugates.

The uptake of 14C-labeled 1-(2-chloroethyl)-1-nitroso-3-(2-hydroxyethyl)urea (HECNU) into the brain was investigated in the rat after intracarotid injection according to the method of OLDENDORF, as well as in cisternal cerebrospinal fluid obtained by suboccipital puncture after i.v. injection of the drug. The brain uptake index was 31.9 +/- 2.9%. Cerebrospinal fluid/blood quotients after i.v. injection were 0.82 at 10 min and 1.10 at 60 min. The results of both methods clearly show that HECNU, in spite of its hydrophilic property, easily penetrates the blood-brain barrier.

A comparative study of in vitro metabolism of isomeric N-nitroso-N-methylaminopyridines (NMPY) by rat liver microsomes revealed remarkable differences in the respective rates of potentially activating and deactivating processes. N-Nitroso-N-methyl-2-aminopyridine (2-NMPY), a potent carcinogen in rats and a mutagen in the Ames test was demethylated to a much greater extent than its non-carcinogenic and non-mutagenic isomers 3-NMPY and 4-NMPY. In contrast to the findings for 2-NMPY, enzymatic denitrosation and N-oxide formation play a substantial role in metabolism of 3-NMPY and 4-NMPY. The respective rates of presumed activating to deactivating metabolic processes were found to be -1.5 for 3-NMPY, 0.5 for 4-NMPY, but 3 for 2-NMPY at 10 mM substrate concentration. At 1 mM concentration (which is more close to the conditions prevailing in carcinogenicity experiments), however, the respective ratio for 2-NMPY was approximately 50. 2-Hydroxypyridine, the hydrolysis product of the putative pyridine-2-diazonium intermediate, generated after oxidative demethylation, was formed in high yields from 2-NMPY. Only traces of 3-hydroxypyridine were formed from 3-NMPY and 4-hydroxypyridine could not be detected at all. Experimental evidence suggests that generation of 2-aminopyridine, which was found in high yields after incubation of 2-NMPY, is explained best by a secondary metabolic process: reductive cleavage of azo coupling products formed by reaction of the pyridine-2-diazonium intermediate with appropriate nucleophiles in the incubation mixture.