Pilar F. Valerón

Assessment of the exposure to organochlorine pesticides, PCBs and PAHs in six species of predatory birds of the Canary Islands, Spain.

In the present study, we investigated the concentrations and distributions of 57 anthropogenic pollutants, including 23 organochlorine pesticides (OCs), 18 polychlorinated biphenyls (PCBs) and 16 polycyclic aromatic hydrocarbons (PAHs) in liver samples from 102 birds of prey of six species that were found dead or that had died during their stay in the Wildlife Recovery Centers of the Canary Islands (Spain) between 2009 and 2012. The dual goal of this work was to determine the occurrence of these contaminants in these six species of birds of prey, and also whether they can be used as bioindicators for monitoring environmental pollution in the region. We found that Accipiter nisus, Falco pelegrinoides and Falco tinnunculus were the most contaminated species. The profiles of contamination among the species were extremely similar in the case of organochlorine contaminants, with DDT and its metabolites as the most abundant compounds. The contamination by DDT and its metabolites, as well as contamination by dieldrin, could be considered high in these animals, much higher than reports from other regions of the planet, which is in agreement with previous reports from our group regarding humans, food and other animals from this area. In contrast, the contamination by PCBs could be considered extremely low and was probably below the threshold of toxicity for these contaminants. The content of carcinogenic/mutagenic PAHs in these animals was clearly dependent on the feeding pattern of the species; however, the levels were also well below the values that were considered toxic in predictive models. This study represents the first report of contamination by PAHs in all these species and is also the first report of PCB levels in Barbary Falcons.

Effects of tamoxifen on serum lipid and apolipoprotein levels in postmenopausal patients with breast cancer

Serum lipids and apolipoprotein levels were measured in twenty postmenopausal women with primary breast cancer, before and three months after tamoxifen therapy (10 mg twice a day). Tamoxifen caused a significant reduction in total serum cholesterol (10%;P < 0.02), and in low-density lipoprotein cholesterol (17%;P < 0.01), and a significant 47 % increase in the subclass 2 of the high density lipoprotein cholesterol (P< 0.01). In addition, tamoxifen caused a 16% increase in apolipoprotein A-I, a 12% decrease in apolipoprotein B (P < 0.05), and a 37% reduction in the serum concentration of lipoprotein(a) (P< 0.01). These results show that tamoxifen brings about an important improvement in serum lipid profile.

Assessment of anticoagulant rodenticide exposure in six raptor species from the Canary Islands (Spain).

Anticoagulant rodenticides are highly toxic compounds that are widely used for pest control of rodents, but that also may threaten the wildlifes health. This work aimed to assess the exposure to first- and second-generation anticoagulant rodenticides (ARs) in six birds of prey species from the Canary Islands (Spain). The concentrations of seven widely used ARs were determined by LC-MS/MS in 104 liver samples of six species of birds of prey (Buteo buteo, Accipiter nisus, Falco pelegrinoides, Falco tinnunculus, Asio otus, and Tyto alba). We determined that 61% of the livers had detectable residues of at least one AR. The most frequently detected AR was bromadiolone, which was detected in 60.3% of the positive cases. The detection frequencies of these compounds varied widely, depending on the species. More than 75% of the A. nisus, T. alba, and A. otus individuals had detectable rodenticide residues in the liver. However, F. tinnunculus exhibited the highest concentrations of AR, with median values above 100 ng/g w.w. We did not detect first-generation ARs in any of the samples. When grouped, nocturnal species exhibited higher AR concentrations than diurnal species (P<0.001). The residue levels were higher among small mammal-eaters than bird-eaters (P<0.01). While most animals exhibited no macroscopic signs of coagulation disorders, approximately 35% exceeded the threshold levels of toxicity, which suggests that these compounds could weaken these animals in their natural environment. In conclusion, the control of rodent populations by ARs suggests that these compounds will enter the food chain and thus threaten the vulnerable populations of raptors on the Canary Islands. Our findings require authorities to ban or strictly control the use of these rodenticides in the natural environment for the conservation of raptors and other predatory species.

Assessment of human health hazards associated with the dietary exposure to organic and inorganic contaminants through the consumption of fishery products in Spain.

In this work we have evaluated the potential carcinogenic and acutely toxic risks associated to the exposure to highly prevalent organic and inorganic contaminants through the consumption of fishery products by the Spanish population. The concentrations of 8 organochlorine pesticides (OCPs), 18 polychlorinated biphenils (PCBs), 7 polycyclic aromatic hydrocarbons (expressed as benzo[a]pyrene toxic equivalents (B[a]Peq)), and three inorganic toxic elements [arsenic (As), cadmium (Cd), and mercury (Hg)] were determined in 93 samples of the most consumed species of white fish, blue fish, cephalopods and seafood species, which were acquired directly in markets and supermarkets in the Canary Islands, Spain. The chemical concentration data were combined with the pattern of consumption of these foodstuffs in order to calculate the daily intake of these contaminants, and on this basis the risk quotients for carcinogenicity and acute toxicity were determined for Spanish adults and children. Our results showed that the daily intake of OCPs, PCBs and B[a]Peq, which is associated to blue fish consumption was the highest within the fish group. The estimated intake of pollutants can be considered low or very low for the individual contaminants, when compared to reference values, except in the case of HCB and As. All the estimated intakes were below the reported Tolerable Daily Intakes. Considering the additive effects of multiple contaminants, the risk of acute toxic effects can also be considered as low or very low. However, our results reflect that the current consumption of white fish in adults and children, and also the blue fish in the case of adults, poses a moderate carcinogenic risk to Spanish consumers, mainly related to their concentrations of As. The conclusions of this research may be useful for the design of appropriate risk communication campaigns.

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER‐2/neu oncogene status and total cellular p185HER‐2 content were simultaneously analyzed in 415 invasive breast‐cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut‐off value of 1.7 for the ratio between the intensity of the HER‐2/neu gene band and the reference gene band, to consider the HER‐2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER‐2 over‐expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER‐2/neu gene amplification. Of these tumors, 87% showed over‐expression of the p185HER‐2. Of the remaining 352 specimens that did not display HER‐2/neu gene amplification, 97% showed no p185HER‐2 over‐expression (p < 0.0001). In 40 selected samples with a p185HER‐2 level lower than 260 fmol/mg protein, the degree of p185HER‐2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER‐2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER‐2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER‐2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER‐2/neu oncogene is amplified or p185HER‐2 is over‐expressed.

Influence of the rehabilitation of injured loggerhead turtles (Caretta caretta) on their blood levels of environmental organic pollutants and elements.

We monitored the levels of 57 organic pollutants and 11 elements in the blood of 61 live stranded loggerhead sea turtles at the beginning and end of the rehabilitation period with the goal of evaluating whether recuperation of normal physiological conditions has an influence on the circulating levels of these pollutants from the blood. According to our results, several pollutants (OCPs, PCBs and toxic elements) are mobilized from storage tissues to the blood in sick turtles, and this is more evident in the subgroup of cachectic animals. We observed a significant decrease in the concentrations of some contaminants at the end of the rehabilitation period, probably due to redistribution processes. In contrast, an increase in the levels of the essential elements selenium and zinc was observed after a period of correct feeding and supplementation during hospitalization.

In vitro evaluation of oestrogenic/androgenic activity of the serum organochlorine pesticide mixtures previously described in a breast cancer case-control study.

Some organochlorine pesticides (OCs) have been individually linked to breast cancer (BC) because they exert oestrogenic effects on mammary cells. However, humans are environmentally exposed to more or less complex mixtures of these organochlorines, and the biological effects of these mixtures must be elucidated. In this work we evaluated the in vitro effects exerted on human BC cells by the OC mixtures that were most frequently detected in two groups of women who participated in a BC case-control study developed in Spain: healthy women and women diagnosed with BC. The cytotoxicity, oestrogenicity, and androgenicity of the most prevalent OC mixtures found in healthy women (H-mixture) and in BC patients (BC-mixture) were tested at concentrations that resembled those found in the serum of the evaluated women. Our results showed that both OC mixtures presented a similar oestrogenic activity and effect on cell viability, but BC-mixture showed an additional anti-androgenic effect. These results indicate that although the proliferative effect exerted by these mixtures on human breast cells seems to depend mainly on their oestrogenic action, the BC-mixture might additionally induce cell proliferation due to its anti-androgenic activity, therefore increasing the carcinogenic potential of this mixture. The findings of this study demonstrate that subtle variations in the composition of a mixture may induce relevant changes in its biological action.

Quantitative analysis of p185HER‐2/neu protein in breast cancer and its association with other prognostic factors

The total cellular p185HER‐2/neu protein (p185) content was measured by ELISA in 346 invasive primary breast cancers, and the results were compared with those of estrogen (ER) and progesterone (PR) receptors, pS2 and Cathepsin D (Cat D) content. At a cut‐off level of 260 fmol/mg protein, 53 of the 346 tumors (15%) were p185‐positive. A significant positive correlation was observed between p185 levels and those of Cat D, and a weaker, though significant, positive correlation with ER, and pS2 levels, but not with those of PR. However, when only the 293 p185‐negative tumors were considered, the correlation between p185 and ER improved substantially, and statistical significance was reached for PR. p185‐positive tumors exhibited lower ER and PR content and higher Cat D content than p185‐negative tumors. The pS2 content, in contrast, did not undergo significant variation. Tumors considered to be p185‐positive were significantly more frequently positive for Cat D at the cut‐off of 45 pmol/mg protein, and were more frequently negative for ER and/or PR, but only significant at the cut‐off of 15 fmol/mg or higher for both steroid receptors. Finally, p185 status was not associated with menopausal status, tumor size, axillary‐lymph‐node invasiveness or distant metastases. These results suggest that 260 fmol/mg protein as the cut‐off for p185 allows the identification of a tumoral sub‐population with a more aggresive phenotype.Int. J. Cancer 74:175–179, 1997.

Consumption of organic meat does not diminish the carcinogenic potential associated with the intake of persistent organic pollutants (POPs)

Numerous studies have shown an epidemiological link between meat consumption and the incidence of cancer, and it has been suggested that this relationship may be motivated by the presence of carcinogenic contaminants on it. Among the most frequently detected contaminants in meat are several types of persistent organic pollutants (POPs), and it is well known that many of them are carcinogenic. On the other hand, an increasing number of consumers choose to feed on what are perceived as healthier foods. Thus, the number of consumers of organic food is growing. However, environmental contamination by POPs is ubiquitous, and it is therefore unlikely that the practices of organic food production are able to prevent this contamination. To test this hypothesis, we acquired 76 samples of meat (beef, chicken, and lamb) of two modes of production (organic and conventional) and quantified their levels of 33 carcinogenic POPs. On this basis, we determined the human meat-related daily dietary exposure to these carcinogens using as a model a population with a high consumption of meat, such as the Spanish population. The maximum allowable meat consumption for this population and the carcinogenic risk quotients associated with the current pattern of consumption were calculated. As expected, no sample was completely free of carcinogenic contaminants, and the differences between organically and conventionally produced meats were minimal. According to these results, the current pattern of meat consumption exceeded the maximum limits, which are set according to the levels of contaminations, and this is associated with a relevant carcinogenic risk. Strikingly, the consumption of organically produced meat does not diminish this carcinogenic risk, but on the contrary, it seems to be even higher, especially that associated with lamb consumption.

Methodology for the Identification of 117 Pesticides Commonly Involved in the Poisoning of Wildlife Using GC–MS-MS and LC–MS-MS

Poisoning with agricultural chemicals is a major threat for wildlife all over the world. We have developed and validated an analytical scheme aimed to the identification and quantification of a wide range of pesticides in fresh liver from wildlife specimens that are routinely delivered to the forensic laboratories for toxicological investigation. The proposed method is comprised of a general solid-liquid extraction followed by purification steps and three complementary liquid or gas chromatographic analyses with triple quadrupole mass spectrometry detection. The developed methodology allows for the determination of 117 highly toxic pesticides in a variety of samples from wildlife poisoning incidents. The validity of the method has been demonstrated in samples from 98 real cases submitted to our laboratory between 2010 and 2012. This method allowed the identification and quantification of poison in 78 of 94 fresh liver samples from wild animals and was successfully used for the identification of pesticides in 35 of 46 non-liver samples. Therefore, the extraction and cleanup method with minor modifications and the potency of triple quadrupole mass spectrometry allow this method to be used to simultaneously detect and quantify or semi-quantify a majority of the most toxic pesticides in a variety of complex and degraded matrices.