Precautionary allergen labelling: perspectives from key stakeholder groups.

Precautionary allergen labelling (PAL) was introduced by the food industry to help manage and communicate the possibility of reaction from the unintended presence of allergens in foods. However, in its current form, PAL is counterproductive for consumers with food allergies. This review aims to summarize the perspectives of all the key stakeholders (including clinicians, patients, food industry and regulators), with the aim of defining common health protection and risk minimization goals. The lack of agreed reference doses has resulted in inconsistent application of PAL by the food industry and in levels of contamination that prompt withdrawal action by enforcement officers. So there is a poor relationship between the presence or absence of PAL and actual reaction risk. This has led to a loss of trust in PAL, reducing the ability of consumers with food allergies to make informed choices. The result has been reduced avoidance, reduced quality of life and increased risk-taking by consumers who often ignore PAL. All contributing stakeholders agree that PAL must reflect actual risk. PAL should be transparent and consistent with rules underpinning decision-making process being communicated clearly to all stakeholders. The use of PAL should indicate the possible, unintended presence of an allergen in a consumed portion of a food product at or above any proposed action level. This will require combined work by all stakeholders to ensure everyone understands the approach and its limitations. Consumers with food allergy then need to be educated to undertake individualized risk assessments in relation to any PAL present.

High levels of dietary fat: alteration of hepatic promutagen activation in the rat.

Male Ola:Sprague-Dawley rats were fed diets containing either low or high levels of fats. After being fed these diets for 4 weeks, the rats were killed and individual hepatic postmitochondrial (S9) fractions were prepared. The ability of these fractions to convert the heterocyclic aromatic amines (HAAs)--2-amino-3-methylimidazo[4,5-f]quinoline; 2-amino-3,4-dimethylimidazo[4,5-f] quinoline; and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (compounds produced during the cooking of proteinaceous food)--to bacterial mutagens was studied, with the use of Salmonella typhimurium TA98 as indicator. Fractions from rats fed high-fat diets exhibited a greater ability to activate the HAAs than did those from rats fed the low-fat diet. The magnitude of the increase was dependent on the type of fat used.

Distribution of radiolabelled [2-14C]IQ and MeIQx in the mouse.

The kinetics of distribution of radiolabelled [2-14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and [2-14C]MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) following the oral administration to BALB/c mice of single doses were studied. Both compounds were taken up into the blood-stream and other tissues rapidly after administration and approximately 20-25% of the radioactive dose of IQ or MeIQx was excreted in urine over 6 h, reflecting the rapid absorption of the mutagens. Significantly greater levels of MeIQx than IQ were isolated from the lungs and blood of treated mice. In studies of the uptake of IQ from closed sections of the gut, little IQ was absorbed from the stomach. Although there was some evidence that it could be absorbed from the large intestine, the primary site of IQ absorption was the small intestine.

Exposure to low concentrations of mutagens alters the mutagenic and lethal effects of 4-nitroquinoline-N-oxide and mitomycin C in Escherichia coli.

Mitomycin C and 4-nitroquinoline-N-oxide are two mutagens which can induce the SOS DNA repair system in Escherichia coli. Growth of E. coli B/r strain WP2, or its uvrA- derivative, in very low concentrations of either mutagen led to the increased sensitization to the lethal and/or mutagenic effects of a subsequent challenge with the same mutagen. These phenomena were not observed in either the recA- or lexA- derivatives of the parent strain. Induction of the adaptive response repair pathway by prior cultivation in low concentrations of N-methyl-N'-nitro-N-nitrosoguanidine reduced both the mutagenic and lethal effects of a subsequent challenge with mitomycin C but not 4-nitroquinoline-N-oxide.

Activation of the food mutagens IQ and MeIQ by hepatic S9 fractions derived from various species.

The ability of hepatic S9 mixes derived from different rodent species (rat, mouse, Syrian and Chinese hamster) to activate the mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was investigated using Salmonella typhimurium strain TA98. In general, the mutagenicity of IQ and MeIQ was greatest in the presence of S9 fractions from Swiss albino mice and least from fractions derived from Chinese hamsters. However, treatment of rats or hamsters with Aroclor 1254 had little or no effect on the activation of IQ or MeIQ to mutagens.

The resistance of E. coli cultivated in low concentrations of dichlorvos to N-methyl-N'-nitro-N-nitrosoguanidine induced mutagenesis.

Cultivation of E. coli B/r strain WP2 in low concentrations of either 4-nitroquinoline N-oxide (4NQO) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) had no effect on the mutagenic or cytotoxic consequences of subsequent challenge with dichlorvos (DCV). However, although the sensitivity of E. coli cells taken from cultures grown in low concentrations of DCV to the effects of 4NQO was unchanged, the cells were more resistant to the mutagenic (but not cytotoxic) consequences of MNNG challenge. This phenomenon was not observed in WP2 derivatives deficient in either error-free (uvrA-) or error-prone (lexA-) DNA-repair, suggesting that a factor common to both these repair pathways may be involved.

Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food.

The ability of 3 plant flavonoids (morin, myricetin and quercetin) and 4 polyphenolic acids (caffeic acid, chlorogenic acid, ellagic acid and ferulic acid) to inhibit the genotoxic effects of a number of cooked-food mutagens (IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2), was investigated in a bacterial mutation assay using Salmonella typhimurium TA98 as indicator and hepatic S9 mixes from either SWR mice or Syrian hamsters as metabolic activating systems. Although the polyphenolic acids failed to have an effect, the flavonoids generally inhibited IQ, MeIQ, MeIQx and Trp-P-1 induced mutagenesis in a dose-dependent manner, irrespective of the source of S9. This was not the case with Trp-P-2 where the flavonoids were only observed to inhibit when SWR mouse S9 but not Syrian hamster S9 was used. Of the 3 compounds, myricetin and quercetin were superior to morin in their inhibitory capacity.

Metabolic conversion of IQ and MeIQ to bacterial mutagens.

The metabolic conversion of 2-amino-3-methyl- and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (IQ and MeIQ respectively) to bacterial mutagens was studied using a bacterial mutation assay. Studies were performed using S9 fractions derived from either corn oil (uninduced) or Aroclor-1254-treated Sprague-Dawley rats. Aroclor 1254 treatment lowered the S9 protein concentration required for optimum levels of mutagenesis, enhanced the numbers of mutants observed and altered the effects of metabolic inhibitors and cofactors added to the assay. Studies with uninduced preparations revealed that IQ and MeIQ exhibited similar responses to the effects of metabolic inhibitors and cofactors involved in detoxication reactions. Both IQ and MeIQ activation appeared to be inhibited by the biogenic amines tryptamine and tyramine and inactivated by conjugation with either acetyl coenzyme A or glutathione.

Exposure of E. coli to nitrosocimetidine induces the adaptive response to alkylating agents.

The cytotoxic and mutagenic properties of nitrosocimetidine (NC), together with its ability to induce the adaptive response DNA-repair pathway were compared with those of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) using Escherichia coli as test organism. MNNG was found to be 250-fold more cytotoxic and 500-fold more mutagenic than NC. Prior cultivation of E. coli in low concentrations of NC protected it against the cytotoxic and/or mutagenic effects of challenge with either NC or MNNG or methyl methanesulphonate (MMS). Induction of the adaptive response by prior cultivation in low concentrations of MNNG reduced the mutagenic and cytotoxic effects of subsequent NC challenge. These results lead us to conclude that although NC is a less potent mutagen than MNNG, the DNA lesions it produces are capable of not only inducing, but also of being repaired by, the adaptive response.

Effect of dietary fibre on the mutagenicity and distribution of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ).

Female BALB/c mice were fed either a fibre-free diet or one supplemented with 30% wheat-bran for 5 weeks. The ability of these mice to convert MeIQ to a bacterial mutagen in vivo was determined using intrasanguinous host-mediated bacterial mutation assays. Less mutagenic activity was detected in the livers of mice fed the bran-supplemented diet compared with those fed the fibre-free diet. Subsequent experiments demonstrated that the effect of brain was not due to modifications in hepatic metabolism, but to changes in uptake of MeIQ from the gastrointestinal tract.

Effect of hepatic cytochrome P-450 inducing agents on mutagen activity in the host-mediated assay.

Intraperitoneal treatment of female BALB/c mice with either phenobarbitone or beta-naphthoflavone led to the induction of various hepatic enzymes associated with xenobiotic metabolism and to increased abilities of hepatic S9 fractions to convert the dietary carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) to an active bacterial mutagen. In the case of another carcinogen, aflatoxin B1 an increase in in vitro hepatic activation was seen only in mice treated with phenobarbitone. In contrast, pretreatment with either phenobarbitone or beta-naphthoflavone reduced the in vivo activity of both aflatoxin B1 and MeIQx in the host mediated bacterial mutation assay. These data indicate that, for some carcinogens at least, the host-mediated assay may be used to predict the carcinogenic consequences of hepatic enzyme induction.

Inhibition of the metabolism of mutagens occurring in food by arachidonic acid.

Hepatic microsomal fractions (microsomes) were prepared from male Sprague-Dawley rats. The effect of arachidonic acid on the conversion of the heterocyclic aromatic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay (indicator: Salmonella typhimurium TA98). Arachidonic acid inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA-repair processes within the bacterial cell. The activation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by this polyunsaturated fatty acid.

Dietary fat modifies the in vivo mutagenicity of some food-borne carcinogens.

Female BALB/c mice were fed a low fat diet (1% safflower oil, by weight) or one supplemented with 25% (by weight) of beef fat or olive oil. The abilities of these diets to modify the in vitro and in vivo hepatic conversion of the dietary carcinogens aflatoxin B1, 2-amino-3, 4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) to bacterial mutagens was evaluated. Dietary olive oil appeared to increase the metabolism of both MeIQ and Trp-P-2 to bacterial mutagens in vivo using the intrasanguineous host-mediated assay. Feeding mice either of the high-fat diets increased hepatic conversion of these two compounds to bacterial mutagens in vitro. Dietary fat had no effect on the metabolism of aflatoxin B1. Subsequent experiments suggested that the in vivo effects of dietary olive oil on MeIQ and Trp-P-2 mutagenesis were due to the induction of hepatic enzyme activities rather than to increased rates of uptake of the carcinogen from the gut-lumen.

Use of continuous culture to study the gastric microflora of a hypochlorhydric patient.

A method of maintaining the microflora obtained from the hypochlorhydric stomach of a patient suffering from hypogammaglobulinaemia has been developed using continuous culture (chemostat) techniques. The culture was maintained at pH 8.0 (the pH of the original gastric juice) and subsequently at pH 7.0 and pH 6.0. Throughout the experiment the total population of the culture remained constant, although the populations of individual bacterial strains varied. Two enzyme parameters, nitrate reductase and beta-glycosidase, were also measured. Optimal enzyme activity was observed at pH 7.0. The results suggest that at the physiological pH values found in hypochlorhydric patients, the gastric flora may play a role in the generation of genotoxins and/or their precursors.

Counteraction of the genotoxicity of some cooked-food mutagens by biogenic amines.

The biogenic amines tryptamine, 5-hydroxytryptamine, tyramine and histamine were assessed for their abilities to modify the genotoxicity of the cooked-food mutagens IQ, MeIQ, MeIQx, Trp-P-1 and Trp-P-2. These measurements were made using a bacterial mutation assay with hepatic fractions from either SWR mice or DSN Syrian hamsters as the activating system and Salmonella typhimurium TA98 as the indicator organism. Although histamine had very little effect on the genotoxicity of these mutagens, the other amines reduced genotoxicity, with tryptamine and 5-hydroxytryptamine exerting the greatest effect. Generally the amines exhibited greater potency when S-9 fractions from mice rather than from hamsters were used.

Effect in vitro of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the hepatic activation of dietary genotoxins by rat post-mitochondrial fractions.

The effect of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid on the conversion of the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to its genotoxic metabolites was investigated using a modified bacterial mutation assay. The assay used Salmonella typhimurium TA98 as an indicator of the mutagenicity and hepatic post-mitochondrial fractions (S-9) from male Sprague-Dawley rats as the activating system. All three fatty acids inhibited the mutagenicity of IQ without effect on the uptake of the active metabolites and/or on the DNA repair processes within the bacterial cell. The activation of three other food mutagens, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and aflatoxin B1 (AFB1) was also inhibited by these fatty acids.

Influence of dietary fat on DNA binding by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in the mouse liver.

Female BALB/c mice were fed either a low (1%)-fat or one of three high-fat diets (containing an additional 25% (w/w) beef fat, hydrogenated vegetable oil or non-hydrogenated vegetable oil) for 4 wk. They were then orally treated with 10 mg 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)/kg body weight and killed 6 hr later. Consumption of the hydrogenated vegetable oil was accompanied by increased DNA adduct formation in mice. The abilities of hepatic S-9 preparations from mice fed the various diets to convert MeIQx to an active bacterial mutagen was assessed using Salmonella typhimurium TA98. Preparations from mice fed the high-fat diets exhibited significantly greater capacity to activate MeIQx than did those from low-fat-fed mice. The greatest increases were seen with S-9 from animals fed either beef fat or hydrogenated vegetable oil.

Caffeine inhibits hepatic-microsomal activation of some dietary genotoxins.

Hepatic microsomal fractions (microsomes) were prepared from female BALB/c mice. The potential of caffeine to modify the ability of microsomes to convert the heterocyclic aromatic amines MeIQ, Trp-P-2 and MeIQx, to bacterial mutagens (indicator: Salmonella typhimurium TA98) was investigated. Caffeine inhibited mutagenicity and did so by inhibiting microsomal metabolism of the three compounds, rather than by altering uptake of the active mutagens and/or interacting with the DNA repair processes in the bacterial cell. Notional Ki values determined for the three heterocyclic amines were similar, suggesting that caffeine inhibits at a stage common to the metabolism of all three compounds.