Antifreeze proteins: characteristics, occurrence and human exposure.

Antifreeze proteins (AFPs), also known as ice structuring proteins, bind to and influence the growth of ice crystals. Proteins with these characteristics have been identified in fish living in areas susceptible to ice formation and in numerous plants and insects. This review considers the occurrence of AFPs and relates it to the likely intake by human populations, with a view to forming a judgment about their safety in foods. Intake of AFPs in the diet is likely to be substantial in most northerly and temperate regions. Much of this intake is likely to be from edible plants, given their importance in the diet, but in some regions intake from fish will be significant. Inadequate data exist to estimate intakes from plants but estimates of intake of AFP from fish are presented for two countries with very different fish consumption, the USA and Iceland. Typical short-term exposure, for instance a portion of cod may contain up to 196 mg AFGP, while the AFP content of the same weight of ocean pout would be up to 420 mg. Average available fish AFP in the diet is calculated to be around 1-10 mg/day in the USA and 50-500 mg/day in Iceland, but these estimates are subject to considerable uncertainty. As far as can be ascertained, AFPs are consumed with no evidence of adverse health effects, either short- or long-term. Given the structural diversity of AFPs, one firm general conclusion that can be drawn from the history of consumption of AFPs is that their functional characteristics do not impart any toxicologically significant effect, in a way that, for instance, a property such as cholinesterase inhibition would. Furthermore, specifically in the case of fish AFPs where some consumption data are available, it is reasonable to infer a lack of allergenicity from the absence of reports of this effect.

The ability of the Comet assay to discriminate between genotoxins and cytotoxins.

The Comet assay has been used widely in genetic toxicology, radiation biology and medical and environmental research. This assay detects single-strand breaks and alkali-labile sites in DNA and DNA degradation due to necrosis or apoptosis. It may also be modified to detect DNA cross-linking. Although a considerable number of chemicals have been tested in the assay there are many aspects of validation to be considered before the method could be considered to provide definitive evidence of genotoxic potential. For example, very few non-genotoxins have been tested to assess specificity of the Comet assay and there has been only one reported study which investigated whether the in vitro Comet assay is prone to false positive responses due to cytotoxicity. We have investigated the response of the alkaline Comet assay in TK6 human lymphoblastoid cells to cytotoxic damage and genotoxic damage. Several compounds which are toxic by different mechanisms were tested in the assay. Cycloheximide and trypsin gave a negative comet response at a highest dose of 5 mg/ml and no toxicity was observed. Sodium lauryl sulphate and potassium cyanide produced a significant increase in DNA migration at cell survival levels of < or = 75%. The distribution of damaged cells indicated that cells at various stages of necrotic cell death were present. Hydrogen peroxide, 4-nitroquinoline oxide, 9-aminoacridine, ethyl methanesulphonate, N-nitroso-N-ethylurea and glyoxal gave a positive comet response. Mitomycin C was negative at survival levels of approximately 70%. These results indicate that the maximum concentration of test substance tested should produce viabilities > 75% in order to avoid false positive responses due to cytotoxicity. The assay was able to detect DNA damage induced by an alkylating agent, an intercalating agent and oxidative damage. The cross-linking agent mitomycin C was not detected if a cut-off point of 75% viability is used as the criterion of a positive response.

Photomutagenicity assays in bacteria: factors affecting assay design and assessment of photomutagenic potential of para-aminobenzoic acid.

Photomutagenicity assays are required for regulatory submissions of some chemicals. As yet there are no well-validated protocols available for these assays. Critical factors which may contribute to the ability of a bacterial assay to detect photomutagens (e.g. dose of UV and test chemical, exposure conditions, light source, bacterial strains) were investigated using two known photomutagens, chlorpromazine and 8-methoxypsoralen. Salmonella typhimurium strains TA98, TA102 and TA1537 and Escherichia coli strains WP2 and WP2(pKM101) were used and differences in the responsiveness of these strains were observed with these substances. Both chemicals were detected using either UV exposure in suspension or on the agar plates. On the basis of these observations and on other results reported in the literature, recommendations are made on protocol aspects for assessing photomutagenic potential in routine screening tests. Using these recommendations the sunscreen para-aminobenzoic acid was tested in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strains WP2 and WP2 (pKM101), using both plate irradiation and suspension exposure conditions. No evidence of mutagenic potential was detected.

Induction of micronuclei in rat bone marrow and peripheral blood following acute and subchronic administration of azathioprine.

The frequency of micronuclei was assessed in polychromatic erythrocytes of bone marrow and in polychromatic and normochromatic erythrocytes in peripheral blood of rats following exposure to azathioprine for 28 days. This was compared with the incidence of micronuclei in bone-marrow following exposure to a single dose of azathioprine. The incidence of micronuclei in bone-marrow polychromatic erythrocytes at the maximum tolerated dose (10 mg/kg) following exposure for 28 days was 29.5/1000. The incidence of micronucleated polychromatic erythrocytes in the peripheral blood at this dose was 4.4/1000. At the maximum tolerated dose in the single-dose study (40 mg/kg) the incidence obtained at 48 h post-treatment was 15.7/1000. This supports the view that the use of animals in a subchronic toxicity study is at least as sensitive for assessing in vivo clastogenic activity as an acute study and could reduce animal usage in toxicology assessments.

Reactions of propylene oxide with 2'-deoxynucleosides and in vitro with calf thymus DNA.

Propylene oxide (PO) is a direct-acting mutagen and rodent carcinogen. We have studied how PO modifies 2'-deoxynucleosides at pH 7.0-7.5 and 37 degrees C for 10 h. PO reacts as an SN2 alkylating agent by forming the following 2-hydroxypropyl (HP) adducts: N6-HP-dAdo (7% yield), 7-HP-Gua (37%) and 3-HP-dThd (4%). Alkylation at N-3 of dCyd resulted in conversion of the adjacent exocyclic imino group at C-4 to an oxygen (hydrolytic deamination) with the formation of a dUrd adduct, 3-HP-dUrd (14%). Ultraviolet spectroscopy and mass spectrometry were used for the structural determination of these adducts. Confirmation of the unexpected 3-HP-dUrd adduct was provided by an accurate mass measurement technique where diagnostic ions in the mass spectra of 3-HP-dUrd were measured to within 0.0005 atomic mass units of the predicted mass. PO was reacted in vitro with calf thymus DNA (pH 7.0-7.5, 37 degrees C, 10 h) and yielded N6-HP-dAdo (1 nmol/mg DNA), 3-HP-Ade (14 nmol/mg DNA), 7-HP-Gua (133 nmol/mg DNA) and 3-HP-dUrd (13 nmol/mg DNA). A mechanism for the hydrolytic deamination of 3-HP-dCyd to 3-HP-dUrd involving the OH on the HP side chain is proposed. This cytosine to uracil conversion may play a role in the mutagenic and carcinogenic activity of this epoxide.

The isolation and characterization of 2-carboxyethyl adducts following in vitro reaction of acrylic acid with calf thymus DNA and bioassay of acrylic acid in female Hsd:(ICR)Br mice.

Reaction of acrylic acid (AA) at pH 7.0 and 37 degrees C for 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo) and thymidine (dThd) resulted in the formation of 2-carboxyethyl (CE) adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (percent yield after 40 days) were 1-CE-dAdo (5%), N6-CE-dAdo (11%) (via Dimroth rearrangement of 1-CE-dAdo), 3-CE-dCyd (7.5%), 7-CE-Gua (4%), 7,9-bis-CE-Gua (0.9%) (formed by reaction of AA with depurinated 7-CE-Gua during the course of the reaction) and 3-CE-dThd (0.5%). The products isolated following in vitro reaction of AA with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-Ade (9.9), N6-CE-Ade (8.2), 7-CE-Gua (7.2) and 3-CE-Thy (1.9). Compound 3-CE-Cyt was not detected. Thus the adducts formed following in vitro reaction of AA with DNA are identical to those formed by in vitro reaction of the carcinogen beta-propiolactone (BPL) with DNA as reported in an earlier paper. Structures were assigned on the basis of identical UV spectra, Rf values on paper chromatograms and Rt values on HPLC as marker compounds prepared from reactions of BPL with 2'-deoxynucleosides and 2'-deoxynucleotides-5'-monophosphoric acids. AA was assayed for carcinogenic activity by s.c. injection (20 mumol, once a week for 52 weeks) in female Hsd: (ICR)Br mice. Two mice with sarcomas at the site of application were observed out of 30 mice. Malignancies were not observed in solvent and no-treatment controls. The bioassay results reported in this paper and elsewhere in the same strain of mice suggest that AA is a weak carcinogen in female Hsd:(ICR)Br mice.

Direct alkylation of 2'-deoxynucleosides and DNA following in vitro reaction with acrylamide.

Reaction of the rodent carcinogen acrylamide (AM) at pH 7.0 and 37 degrees C for 10 and 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), and thymidine (dThd) resulted in the formation of 2-formamidoethyl and 2-carboxyethyl adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (% yield after 40 days) were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo) (8%), N6-CE-dAdo (21%) (via Dimroth rearrangement of 1-CE-dAdo), 1-CE-dGuo (4%), 7-(2-formamidoethyl)-Gua (7-FAE-Gua) (6%), 7, 9-bis-FAE-Gua (1%) (formed by reaction of AM with depurinated 7-FAE-Gua during the course of the reaction), and 3-FAE-dThd (4%). The products isolated following in vitro reaction of AM with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-dAdo (5.5), N6-CE-dAdo (1.4), 3-CE-dCyd (2.8), 1-CE-dGuo (0.3), and 7-FAe-Gua (1.6). Compound 3-FAE-dThd was not detected. Structures were assigned on the basis of chemical properties, UV spectra, and electron impact, chemical ionization, desorption chemical ionization, Californium-252 fission fragment ionization, and fast atom bombardment mass spectra. A facile hydrolysis of the amide group to a carboxylic acid was observed when AM alkylated a ring nitrogen adjacent to an exocyclic nitrogen atom. In previous studies, we had observed an analogous phenomenon when studying the in vitro reactions of acrylonitrile with DNA, i.e., a facile hydrolysis of nitrile to carboxylic acid when acrylonitrile alkylated (via Michael addition) a ring nitrogen adjacent to an exocyclic nitrogen. Since the nitrile group hydrolyzes to a carboxylic acid via an amide intermediate, we had hypothesized in the present study that the same facile hydrolysis of amide to carboxylic acid would occur under identical stereochemical conditions as had occurred with the nitrile group. Thus, in vitro alkylation of calf thymus DNA by both acrylonitrile and, in the present study, AM, resulted in mixed adduct formation.