C. Galli

Threshold of toxicological concern for chemical substances present in the diet: A practical tool for assessing the need for toxicity testing

The de minimis concept acknowledges a human exposure threshold value for chemicals below which there is no significant risk to human health. It is the underlying principle for the US Food and Drug Administration (FDA) regulation on substances used in food-contact articles. Further to this, the principle of Threshold of Toxicological Concern (TTC) has been developed and is now used by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in their evaluations. Establishing an accepted TTC would benefit consumers, industry and regulators, since it would preclude extensive toxicity evaluations when human intakes are below such threshold, and direct considerable time and cost resources towards testing substances with the highest potential risk to human health. It was questioned, however, whether specific endpoints that may potentially give rise to low-dose effects would be covered by such threshold. In this review, the possibility of defining a TTC for chemical substances present in the diet was examined for general toxicity endpoints (including carcinogenicity), as well as for specific endpoints, namely neurotoxicity and developmental neurotoxicity, immunotoxicity and developmental toxicity. For each of these endpoints, a database of specific no-observed-effect levels (NOELs) was compiled by screening oral toxicity studies. The substances recorded in each specific database were selected on the basis of their demonstrated adverse effects. For the neurotoxicity and developmental neurotoxicity databases, it was intended to cover all classes of compounds reported to have either a demonstrated neurotoxic or developmentally neurotoxic effect, or at least, on a biochemical or pharmacological basis were considered to have a potential for displaying such effects. For the immunotoxicity endpoint, it was ensured that only immunotoxicants were included in the database by selecting most of the substances from the Luster et al. database, provided that they satisfied the criteria for immunotoxicity defined by Luster. For the developmental toxicity database, substances were selected from the Munro et al. database that contained the lowest NOELs retrieved from the literature for more than 600 compounds. After screening these, substances showing any effect which could point to developmental toxicity as broadly defined by the US were recorded in the database. Additionally, endocrine toxicity and allergenicity were addressed as two separate cases, using different approaches and methodology. The distributions of NOELs for the neurotoxicity, developmental neurotoxicity and developmental toxicity endpoints were compared with the distribution of NOELs for non-specific carcinogenic endpoints. As the immunotoxicity database was too limited to draw such a distribution of immune NOELs, the immunotoxicity endpoint was evaluated by comparing immune NOELs (or LOELs-lowest-observed-effect levels-when NOELs were not available) with non-immune NOELs (or LOELs), in order to compare the sensitivity of this endpoint with non-specific endpoints. A different methodology was adopted for the evaluation of the endocrine toxicity endpoint since data currently available do not permit the establishment of a clear causal link between endocrine active chemicals and adverse effects in humans. Therefore, this endpoint was analysed by estimating the human exposure to oestrogenic environmental chemicals and evaluating their potential impact on human health, based on their contribution to the overall exposure, and their estrogenic potency relative to endogenous hormones. The allergenicity endpoint was not analysed as such. It was addressed in a separate section because this issue is not relevant to the overall population but rather to subsets of susceptible individuals, and allergic risks are usually controlled by other means (i.e. labelling) than the Threshold of Toxicological Concern approach. (ABSTRACT TRUNCATED)

Patulin in apple-based foods: occurrence and safety evaluation

Patulin is a mycotoxin produced by certain species of Penicillium and Aspergillus, often detectable in mouldy fruits and their derivatives. On the basis of a PMTDI of 0.4 μg/kg bw, limit values of 50 μg/kg or 50 μg/l of patulin have been set in fruit derivatives. To estimate the quantity of patulin that can be taken in with the diet, we analysed by HPL C samples of apples and apple derivatives which are most likely to be contaminated with patulin. In apple juices and in homogenized babyfoods, the mycotoxin concentration was always below the established limits, while in some samples of juice with pulp the mycotoxin content exceeded the safe levels. In rotten apples, not only was the amount of patulin extraordinarily high in the rotten area, but the mycotoxin had also spread to the part unaffected by mould. The data presented in this study indicate that the intake of patulin with apple derivatives is usually below the tolerable level of 0.4 μg/kg bw/day, but since the patulin content in apples can vary considerably, the quality of fruits used in the production of apple derivatives should be strictly controlled in order not to exceed the safe limits.

Cytokines and irritant contact dermatitis

Skin irritation is a complex phenomenon that involves resident epidermal cells, fibroblasts of dermis, and endothelial cells as well as invading leukocytes interacting with each other under the control of a network of cytokines and lipid mediators. Keratinocytes play an important role in the initiation and perpetuation of skin inflammatory reactions through the release of, and responses to cytokines. While resting keratinocytes produce some cytokines constitutively, a variety of environmental stimuli, such as tumor promoters, ultraviolet light and chemical agents, can induce epidermal keratinocytes to release inflammatory cytokines (IL-1, TNF-alpha), chemotactic cytokines (IL-8, IP-10), growth promoting cytokines (IL-6, IL-7, IL-15, GM-CSF, TGF-alpha) and cytokines regulating humoral vs. cellular immunity (IL-10, IL-12, IL-18). The role of cytokines in xenobiotics-induced skin irritation and the early molecular events that follow the treatment with irritant compounds will be discussed.

DIFFERENTIATION OF DOPAMINERGIC AND NORADRENERGIC NEURONS IN RAT SPINAL CORD

Abstract— Norepinephrine (NE), dopamine (DM) and 3‐methoxy‐4‐hydroxyphenylacetic acid (HVA) content have been measured in different parts of rat spinal cord and cerebellum by a gas chromatographic mass spectrometric method. In cerebellum, which does not contain dopaminergic neurons, the ratio of NE to DA content was 47, whereas in parts of the spinal cord this ratio varied between 11 and 19. In the cord after desipramine (25 mg/kg, i.p.) plus 6‐hydroxydopamine (6‐HDA, 100/jg intracisternally), there was a significant depletion of DM but not of NE. Conversely, after benztropine (25 mg/kg, i.p.) plus 6‐HDA there was a significant depletion of NE but not of DM. Chlorpromazine (10 mg/kg, i.p.) or clozapine (25 mg/kg, i.p.) caused a significant increase in spinal cord HVA concentration 1 h after treatment. Evidence is presented which suggests that the increased HVA measured in the cord did not originate in the brain. After electrolytic lesion of the locus coeruleus there was a significant reduction of NE but not of DM. Spinal cord DM and NE were depleted by reserpine in a dose‐dependent manner, the threshold dose for DM depletion being less than that for NE depletion. Seven days after cord transection at T10 spinal cord DM was significantly reduced in the lumbar region. These results suggest that dopaminergic neurons exist in rat spinal cord independently of noradrenergic neurons and that the DM is likely to be present in the terminals of descending axons.

Significance of dopamine metabolites in the evaluation of drugs acting on dopaminergic neurones

The effect of various drugs was studied on 3-methoxytyramine (3-MT) concentrations in rat striatum. The drugs were chosen for their ability to interfere with the dopaminergic system at different levels. Dopamine (DA) acidic metabolites, i.e. homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC), were also measured. Changes of 3-MT, unlike those of DOPAC and HVA, seem to reflect the functional activity of dopaminergic neurons. In fact drugs believed to increase or decrease DA content in the synaptic cleft produce predictable changes of striatal 3-MT. Thus cocaine, nomifensine and d-amphetamine increase 3-MT concentrations while gamma-butyrolactone, alpha-methyltyrosine and apomorphine decrease it.

Cross-reactivity between milk proteins from different animal species

Cows milk allergy is quite frequent in the first years of human life. When breast‐feeding is not possible, a cows milk substitute must be provided for allergic subjects. Different alternatives to cows milk have been suggested as protein sources (soy, hydrolysed proteins, goats milk, etc.), but all these dietetic solutions are not without risks for polyallergic or more sensitive subjects.

Cross-reactivity between mammalian proteins

BACKGROUND Cross-reactivity between food allergens occurs when they share part of their amino acid sequence, or when their three-dimensional molecular structure causes them to have a similar capacity to bind specific antibodies. OBJECTIVES To review data from our laboratory on cross-reactivity between mammalian proteins (milk and meat allergens). METHODS Studies used immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/polyacrylamide gel electrophoresis and immunoblotting), and animal monoclonal antibodies. RESULTS The findings suggest that animal monoclonal antibodies specific for cows milk proteins are able to recognize the major part of milk proteins from mammals bred in Mediterranean countries (sheep, goat, and buffalo); weak cross-reactivity was observed with milk proteins from mares and donkeys. None of the antibodies used in our studies reacted with proteins from an exotic mammalian species: the camel. Similar cross-reactions were found with human circulating immunoglobulin E from children allergic to milk. With regard to beef allergy, monoclonal antibodies specific for bovine serum albumin cross-reacted only with ovine serum albumin, whereas the number of sera from allergic children able to recognize other mammalian serum albumins depended directly on the closeness of phylogenetic relationship between animal species and inversely on the percent identity with human serum albumin in the main epitopic sequence. CONCLUSION An area of heterogeneity between animal and human species in a critical amino acid sequence (epitope) of an allergen can determine the degree of immunogenic activity.

Epidermal cytokines in experimental contact dermatitis.

Topical exposure to a variety of xenobiotics may result in irritant as well as allergic contact dermatitis both in rodents and in humans. Despite their induction by different mechanisms, they cannot be differentiated by macroscopic appearance and, by histological examination they are both generally characterized by a perivascular mononuclear cell infiltrate and capillary hyperpermeability. Recently, cytokines, a family of inducible glycoproteins that play a pivotal role in immune and inflammatory reactions, have been identified as useful tools for differentiation of irritant and allergic contact dermatitis. In this article the role of cytokines in the development and differentiation of irritant and allergic contact dermatitis is discussed.

Differential effects of dietary fatty acids on the accumulation of arachidonic acid and its metabolic conversion through the cyclooxygenase and lipoxygenase in platelets and vascular tissue.

Semisynthetic diets containing either corn oil (CO) or butter (B) (11 and 2.2 en % as linoleic acid, respectively) were fed to male rabbits for periods of 3 weeks and 3 months. The CO diet, in respect to the B diet, induced higher levels of linoleic acid (LA) and lower levels of arachidonic acid (AA) in platelet phospholipids, lower levels of AA in aortic phosphatidylinositol (PI) and accumulation of both LA and AA in liver lipids. The thresholds for aggregation with AA, but not with collagen, were higher in the CO group and the formation of thromboxane B2 (TXB2) from [14C] AA, but not from endogenous substrate after collagen stimulation, was lower in the same group. Formation of PGE2-like material by incubated aortas was higher in the B group. In the CO group, platelet cyclooxygenase appeared to be selectively depressed. The correlations among diet-induced fatty acid changes in platelet and aortic lipids, platelet aggregation and thromboxane and prostacyclin formation are discussed.

Use of IL-18 production in a human keratinocyte cell line to discriminate contact sensitizers from irritants and low molecular weight respiratory allergens

Assessment of allergenic potential of chemicals is performed using animal models, such as the murine local lymph node assay, which does not distinguish between respiratory and contact allergens. Progress in understanding the mechanisms of skin sensitization, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitization testing. The aim of the present study was to evaluate the possibility to use intracellular interleukin-18 (IL-18) production to assess in vitro the contact sensitization potential of low molecular weight chemicals. The human keratinocyte cell line NCTC2455 was used. Cells were exposed to contact allergens (cinnamaldehyde, dinitrochlorobenzene, glyoxal, isoeugenol, p-phenylediamine, resorcinol, tetramethylthiuram disulfide, 2-mercaptobenzothiazole, 4-nitrobenzylbromide), to proaptens (cinnamyl alcohol, eugenol), to respiratory allergens (diphenylmethane diisocyanate, trimellitic anhydride, ammonium hexachloroplatinate) and to irritants (sodium lauryl sulphate, salicylic acid, phenol). Cell associated IL-18 were evaluated 24 later. At not cytotoxic concentrations (cell viability higher of 75%, as assessed by MTT reduction assay), all contact sensitizers, including proaptens, induced a dose-related increase in IL-18, whereas both irritants and respiratory failed. Similar results were also obtained using primary human keratinocytes. Results were reproducible, and the method could be transferred to another laboratory, suggesting the potential use of the test in immunotoxicity testing strategies. Overall, results obtained indicated that cell-associated IL-18 may provide an in vitro tool for identification and discrimination of contact versus respiratory allergens and/or irritants.