Coatings in food contact materials: Potential source of genotoxic contaminants?

Up till now, no harmonized EU regulation exists on chemicals used in coatings for food contact materials (FCM). Therefore, these substances need to comply with the general provisions of EU Regulation 1935/2004 and, if present, with national legislation. Different 'inventory lists' of compounds that might be present in coatings are available, but for hundreds of these substances, the potential human health impact of their use in FCM coatings has not (recently) been evaluated. Since detailed evaluation of all compounds is not feasible, a pragmatic approach was developed to identify substances with a potential concern for human health. First, an inventory was assembled containing all substances potentially used in coatings. Afterwards, the genotoxic potential of the non-evaluated substances was predicted in silico using two structure-activity relationship (SAR) software programs. For substances yielding structural alerts in both models, genotoxicity data were collected from previous European evaluations in a non-FCM context and from the European CHemicals Agency (ECHA) website. In total, 53 substances were identified as genotoxic in both in silico models, of which ten were considered to be of high concern. For most of the substances, additional toxicological information is needed.

Genetic damage in humans exposed to extremely low-frequency electromagnetic fields.

The classification of extremely low-frequency magnetic fields by the International Agency for Research on Cancer in the group of 'possible human carcinogens' (group 2B) is essentially based on epidemiologic evidence showing an association between MF exposures and childhood leukaemia. Despite many in vitro and in vivo investigations, there is no established causal relationship yet. However, human cytogenetic biomonitoring studies that were conducted in the past show predominantly positive results, i.e. increased cytogenetic damage in peripheral blood lymphocytes or buccal cells of ELF-MF-exposed subjects. This is important given the established link between observed cytogenetic damage in cells of people and an increased cancer risk. We here conducted an evaluation of the published investigations and found that many of the studies clearly have shortcomings, which often prevent any firm conclusion. As a matter of fact, there are reasons to believe that effects are not that impressive. However, the totality of the studies cannot simply be disregarded warranting further caution and the application, to a certain extent, of the precautionary principle.

Investigation into the genotoxicity of water extracts from Hypoxis species and a commercially available Hypoxis preparation.

We performed an in vitro evaluation of the genotoxic potential of water extracts from four Hypoxis species (Hypoxis hemerocallidea, H. colchicifolia, H. rigidula, H. acuminata) and a commercial preparation thereof using the neutral red uptake (NRU) assay, the alkaline comet assay and the cytome assay in human hepatoma HepG2 cells. The relative cytotoxicity of these samples was established by determining their NI50 values (50% inhibition of NRU), and these results were used for dose-finding in genotoxicity tests. None of the tested extracts were identified as genotoxic in both the alkaline comet assay and cytome assay.

Testing chemical agents with the cytokinesis-block micronucleus cytome assay.

In order to evaluate the applicability of the cytokinesis-block micronucleus cytome assay in routine mutagenicity testing we investigated with this method different chemicals having different mechanisms of action: non-mutagens, direct-acting basealtering mutagens, direct-acting cross-linking mutagens, clastogens including a radiomimetic chemical, indirect-acting spindle poisons and indirect-acting enzyme inhibitors. We looked at the presence of micronuclei as biomarkers for either the loss of chromosome fragments (clastogen) or the loss of a whole chromosome (aneugen), nucleoplasmic bridges as biomarkers for complex rearrangements (e.g., dicentric chromosomes) and nuclear buds as biomarkers for gene amplification. The cytome assay proved to be a suitable tool to investigate genetic effects of environmental agents and to provide insight into their working mechanisms as all chemicals tested showed the expected response.

1,4-diarylpiperazines and analogs as anti-tubercular agents: synthesis and biological evaluation.

Despite progress in modern chemotherapy to combat tuberculosis, the causative pathogen Mycobacterium tuberculosis (M.tb.) is far from eradicated. Bacillary resistance to anti-mycobacterial agents, bacillary persistence and human immunodeficiency virus (HIV) co-infection hamper current drug treatment to completely cure the infection, generating a constant demand for novel drug candidates to tackle these problems. A small library of novel heterocyclic compounds was screened in a rapid luminometric in vitro assay against the laboratory M.tb. strain H37Rv. A group of amidines was found to have the highest potency and was further evaluated for acute toxicity against C3A hepatocytes. Next, the most promising compounds were evaluated for activity against a multi-drug resistant clinical isolate. The group of amidines was also tested for their ability to kill intracellular M.tb. residing in mouse J774A.1 macrophages. Finally, we report on a correlation between the structural differences of the compounds and their anti-mycobacterial activity.

In vitro and in vivo genotoxicity of radiofrequency fields.

There has been growing concern about the possibility of adverse health effects resulting from exposure to radiofrequency radiations (RFR), such as those emitted by wireless communication devices. Since the introduction of mobile phones many studies have been conducted regarding alleged health effects but there is still some uncertainty and no definitive conclusions have been reached so far. Although thermal effects are well understood they are not of great concern as they are unlikely to result from the typical low-level RFR exposures. Concern rests essentially with the possibility that RFR-exposure may induce non-thermal and/or long-term health effects such as an increased cancer risk. Consequently, possible genetic effects have often been studied but with mixed results. In this paper we review the data on alleged RFR-induced genetic effects from in vitro and in vivo investigations as well as from human cytogenetic biomonitoring surveys. Attention is also paid to combined exposures of RFR with chemical or physical agents. Again, however, no entirely consistent picture emerges. Many of the positive studies may well be due to thermal exposures, but a few studies suggest that biological effects can be seen at low levels of exposure. Overall, however, the evidence for low-level genotoxic effects is very weak.

Mutagenic and antimutagenic properties of extracts from South African traditional medicinal plants.

AIM OF THE STUDY: The aim of this paper was to summarize the results of our investigations on the in vitro genotoxic as well as antigenotoxic effects of a great number of selected South African traditional medicinal plants. MATERIALS AND METHODS: Investigations of methanol and dichloromethane extracts of selected plants were conducted with the bacterial Ames, Umu-C and VITOTOX tests, and with the cytochalasin B micronucleus test and alkaline comet assay in human white blood cells. RESULTS: A number of extracts were found to have genotoxic properties. Amongst the genotoxic plant extracts, especially methanol extracts of Helichrysum simillimum DC. (Asteraceae) should be highlighted. On the other hand, some plant extracts also showed antimutagenic potential. Here Bauhinia galpinii N.E.Br. (Fabaceae) and especially Chlerodendrum myricoides (Hochst.) Vatke (=Rotheca myricoides (Hochst.) Steane & Mabb.; Lamiaceae) appear to have antimutagenic properties. CONCLUSION: The safe use of Helichrysum similimum should be questioned and further investigations on its mutagenicity and overall biological properties should be encouraged. Antimutagenic properties of especially Bauhinia galpinii and Rotheca myricoides are considered of particular interest as it may be assumed that these antimutagenic natural substances are able to lower the cancer risk from everyday exposures to environmental mutagens as well as to mutagenic pharmaceuticals.

Nucleotide excision repair impairment by nodularin in CHO cell lines due to ERCC1/XPF inactivation.

The problem of toxicity of cyanobacterial toxins is of increasing concern, as the incidence of such blooms grows. Among the toxins, the most abundant in the environment are hepatotoxins known as nodularins and microcystins. These toxins are responsible for almost all known cases of fresh and brackish water intoxication and are responsible for recurrent episodes of human and animal illness and death. Moreover, they are believed to be potent tumor promoters and initiators. However, the mechanisms by which these toxins induce liver cancer are not well understood. The aim of the present study was to determine the effect of nodularin on the kinetics of nucleotide excision repair (NER) in Chinese hamster ovary (CHO) cells exposed to UV radiation. The first set of experiments was performed to define the optimal treatment conditions for nodularin to avoid the possibility of encountering false positive signals in the comet assay due to the apoptogenic activity of nodularin. Based on the analysis of apoptosis, the 6-h treatment time of cells with nodularin (1mug/ml, 10mug/ml and 20mug/ml) was chosen for the alkaline comet assay. The kinetics of NER was determined in CHO cell lines: AA8 (wild-type) and mutant cell lines: UV135 (XPG(-)), UV41 (XPF(-)) and UV20 (ERCC1(-)) exposed to 20J/m(2) UV radiation. The micronucleus assay was performed to determine a residual DNA damage in four cell lines treated with nodularin (10mug/ml) and exposed to equitoxic doses UV radiation. Radiation doses of UV producing 50% of survival for AA8, UV135, UV20 and UV41 cell lines were calculated from UV survival curves. The results show that nodularin impairs the incision/excision step of NER in CHO cells by the ERCC1/XPF inactivation and leads to an increased level of UV-induced cytogenetic DNA damage.

Automated detection of irradiated food with the comet assay.

Food irradiation is the process of exposing food to ionising radiation in order to disinfect, sanitise, sterilise and preserve food or to provide insect disinfestation. Irradiated food should be adequately labelled according to international and national guidelines. In many countries, there are furthermore restrictions to the product-specific maximal dose that can be administered. Therefore, there is a need for methods that allow detection of irradiated food, as well as for methods that provide a reliable dose estimate. In recent years, the comet assay was proposed as a simple, rapid and inexpensive method to fulfil these goals, but further research is required to explore the full potential of this method. In this paper we describe the use of an automated image analysing system to measure DNA comets which allow the discrimination between irradiated and non-irradiated food as well as the set-up of standard dose-response curves, and hence a sufficiently accurate dose estimation.

Cytogenetic analysis of tannery workers in Morocco.

This study investigated the possible genetic effects in blood lymphocytes of tannery workers from Morocco being professionally exposed to multiple chemical agents. It was shown that the frequencies of cells with chromosome aberrations and micronuclei were significantly increased in the lymphocytes of the workers compared with the frequencies found in an unexposed control population.

Cytogenetic biomonitoring of the Mzamza population exposed to untreated wastewaters.

A cytogenetic biomonitoring study was performed on people from the Mzamza community near Settat, Morocco. These subjects live in and near the Bou Moussa valley where wastewaters from a great number of industries are discharged without any treatment. This wastewater is used as a source of drinking water for their cattle and irrigation of their land. The Mzamza population is therefore presumably exposed to continuous low doses of different kinds of pollutants. Our study demonstrated significant increases in micronucleated white blood cells indicating a considerable genetic risk in these subjects.

Seasonal variations in spontaneous levels of DNA damage; implication in the risk assessment of environmental chemicals.

The alkaline comet assay was used to investigate DNA damage levels in white blood cells of 45 normal healthy subjects. Therefore blood was sampled at four different periods, namely in February, June, August and November of the same year. Higher DNA damage levels were found in summertime, as well as higher levels of 1-hydroxypyrene in urine in this period. This suggests a higher exposure to polycyclic hydrocarbons in the summer compared with other periods of the year. The observed seasonal variation in DNA damage levels is in agreement with some, but in contradiction with other data. Seasonal variations in DNA damage levels can easily be explained by the existence of different confounders that may influence the results of a biomonitoring study. Besides sunlight and environmental pollution, also diet, allergy and physical exercise, for example, were already identified as important influencing factors. The investigation confirms that the blood sampling period is crucial in the planning and interpretation of biomonitoring studies.

A battery of DNA effect biomarkers to evaluate environmental exposure of Flemish adolescents.

The present paper deals with the evaluation of a battery of genotoxicity biomarkers in healthy Flemish adolescents and their relation with common pollutants occurring in their life environment. DNA damage as reflected by the comet assay appeared to be most sensitive to ozone (partial r(2) = 0.102, p < 0.00001), and to a lesser extent to ortho-cresol (partial r(2) = 0.055; p = 0.001) and 1-hydroxy-pyrene (1-OH-pyrene, partial r(2) = 0.031; p = 0.013). 8-hydroxy-deoxyguanosine (8-OHdG) was only related to ortho-cresol (r(2) = 0.069; p < 0.007). Interestingly, the comet assay results and urinary 8-OHdG concentrations were positively correlated with a Pearson r = 0.21 (p = 0.003, N = 200). Logistic regression models revealed significant relations between chromatid breaks and 1-OH-pyrene (relative risk (RR): 1.58; p = 0.008), and t,t-muconic acid (RR: 1.71; p = 0.014). There was no correlation between micronucleus formation or occurrence of chromosomal or chromatid breaks on the one hand and comet or 8-OHdG results on the other hand. Thus, in this study the comet assay on whole blood samples and urine 8-OHdG measurements especially appeared sensitive biomarkers for assessing the genetic effects of environmental pollutants to which adolescents may be exposed.

Investigation of co-genotoxic effects of radiofrequency electromagnetic fields in vivo.

We investigated the possible combined genotoxic effects of radiofrequency (RF) electromagnetic fields (900 MHz, amplitude modulated at 217 Hz, mobile phone signal) with the drinking water mutagen and carcinogen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Female rats were exposed to RF fields for a period of 2 years for 2 h per day, 5 days per week at average whole-body specific absorption rates of 0.3 or 0.9 W/kg. MX was given in the drinking water at a concentration of 19 microg/ml. Blood samples were taken at 3, 6 and 24 months of exposure and brain and liver samples were taken at the end of the study (24 months). DNA damage was assessed in all samples using the alkaline comet assay, and micronuclei were determined in erythrocytes. We did not find significant genotoxic activity of MX in blood and liver cells. However, MX induced DNA damage in rat brain. Co-exposures to MX and RF radiation did not significantly increase the response of blood, liver and brain cells compared to MX exposure only. In conclusion, this 2-year animal study involving long-term exposures to RF radiation and MX did not provide any evidence for enhanced genotoxicity in rats exposed to RF radiation.

Evaluation of the mutagenic and antimutagenic effects of South African plants.

Dichloromethane and 90% methanol extracts of 42 South African plants were screened for mutagenicity and antimutagenicity using the Salmonella/microsome mutagenicity assay (Ames) against Salmonella typhimurium TA98 and TA100 bacterial strains in the presence and absence of metabolic activator S9. The methanol extracts from whole plants of Helichrysum simillimum, Helichrysum herbaceum and Helichrysum rugulosum indicated mutagenicity. These are the first reported tests on the mutagenicity of Helichrysum species. Six species indicated antimutagenic properties, all in the presence of S9: methanol leaf extract of Bauhinia galpinii, and dichloromethane leaf extracts of Bauhinia galpinii, Clerodendrum myricoides, Datura stramonium, Buddleja saligna, Millettia sutherlandii and Sutherlandia frutescens.

Genotoxicity of the Musi River (Hyderabad, India) investigated with the VITOTOX test.

The bacterial VITOTOX genotoxicity test was used to screen water samples collected from three different stations along the banks of the river Musi, in Hyderabad, India. Water was collected at three stations that differed from each other in the nature of the surrounding industrial and other activities. A number of different pollutants were also measured in water, soil and air samples. The three stations were found highly polluted and different with regard to the genotoxicity and toxicity of their samples. These results demonstrate the need for further biological studies in this area to generate valuable data on genomic instability, risk assessment of cancer, and to provide avenues for risk management.

Genetic effects of radiofrequency radiation (RFR).

The possible effects of radiofrequency (RF) exposure on the genetic material of cells are considered very important since damage to the DNA of somatic cells can be linked to cancer development or cell death whereas damage to germ cells can lead to genetic damage in next and subsequent generations. This is why the scientific literature reports many investigations on the subject. According to a number of review papers, the conclusion so far is that there is little evidence that RFR is directly mutagenic and that adverse effects that were reported in some of the papers are predominantly the result of hyperthermia. Yet, some subtle indirect effects on DNA replication and/or transcription of genes under relatively restricted exposure conditions cannot be ruled out. Furthermore, the possibility of combined effects of RFR with environmental carcinogens/mutagens merits further attention. The present paper takes into account more recent investigations but the conclusion remains the same. A majority of studies report no increased (cyto)genetic damage but yet, a considerable number of investigations do. However, many studies were not sufficiently characterized, are therefore difficult to replicate and cannot be compared to others. Experimental protocols were very different from one study to another and investigations from a single laboratory were very often limited in the sample size or number of cells investigated, preventing a robust statistical analysis. Subtle, but significant differences between RFR-exposed and sham-exposed cells cannot be found in such conditions. For the above reasons, it was concluded at a workshop in Löwenstein (November 2002) that further investigations by individual laboratories most probably will not add much to the discussion of radiofrequency radiation (RFR) genotoxicity. Large, well coordinated, international collaborative studies involving participation of several experienced scientists are considered an alternative of uttermost importance. One such study is now being planned.

Cytogenetic investigation of workers professionally exposed to phosphates and their derivatives.

The phosphor industry in Morocco employs a great number of workers that may be exposed to this essential but also toxic compound. In the present investigation, professionally exposed subjects from two different production lines (miners and production unit workers) were investigated for the presence of genetic damage in their peripheral blood lymphocytes. This was done with the well-known micronucleus assay. The proliferation and mitotic indices were also investigated. It was found that the micronucleus frequency was considerably increased compared to the frequency in non-exposed control subjects. Mitotic and proliferation indices were not increased, at least not in a statistically significant way. At the time being this investigation should be considered as a preliminary study in which the influence of potential confounders cannot be adequately assessed. However, our results are non-equivocal and clearly indicate a potential health risk in these workers.

Investigation of the antimutagenic effects of selected South African medicinal plant extracts.

Dichloromethane extracts from different parts of Rhamnus prinoides, Ornithogalum longibracteatum, Gardenia volkensii, Spirostachys africana, Diospyros whyteana, Syzigium cordatum and Prunus africana were investigated for mutagenic and antimutagenic effects in Salmonella/microsome and micronucleus tests. None of the extracts tested in the Ames test were found to induce mutations or to modify the effect of the mutagen 4-nitroquinoline-oxide (4NQO). In the micronucleus test, extracts from twigs/bark of R. prinoides, twigs of D. whyteana, P. africana and S. cordatum significantly lowered the effect of the mutagen mitomycin C (MMC). Extracts from twigs/bark of G. volkensii and S. africana were genotoxic in the micronucleus test, while extracts of O. longibracteatum leaves potentiated the genotoxicity of MMC. This preliminary investigation shows that plant extracts used in traditional medicine may have particular effects with regard to mutagenicity and antimutagenicity indicating careful use in some instances and the need to isolate their active principles for further research.