Marta S. Monteiro

Assessment of biomarkers of cadmium stress in lettuce.

Laboratory and field studies have provided encouraging insights into the capacity of plants to act as biomonitors of environmental quality through the use of biomarkers. However, a better understanding of the overall process of Cd-induced senescence, describing the cascade of Cd effects in plants is needed for a selection of relevant biomarkers of Cd stress. In order to approach this, 5-week old Lactuca sativa L. were exposed for 14 days to 100muM Cd(NO(3))(2) and harvested at days 0, 1, 3, 7 and 14. The parameters measured included classical endpoints (shoot and root growth) and biochemical endpoints related to photosynthesis, nutrients content, and oxidative stress. Cadmium-exposed plants displayed nutrient imbalances in leaves and roots. Photosynthetic efficiency was significantly decreased and lipid peroxidation was enhanced. Antioxidant enzymes were significantly altered during exposure-catalase was inhibited by the end of exposure and peroxidase was induced at day 1 in young leaves. These alterations culminated in a decrease in shoot growth after 14-days exposure to Cd. Biochemical alterations could be used in integrative approaches with classical endpoints in ecotoxicological tests for Cd and after further testing in real scenarios conditions, they could form the basis of a plant biomarkers battery for monitoring and predicting early effects of exposure to Cd.

Synergistic effects caused by atrazine and terbuthylazine on chlorpyrifos toxicity to early-life stages of the zebrafish Danio rerio

This study examined the effects of three widely used pesticides that have been previously detected in aquatic systems neighbouring agricultural fields on the early-life stages of the zebrafish Danio rerio. Tests involving single exposures and binary combinations of the s-triazine herbicides (atrazine and terbuthylazine) and the organophosphate insecticide chlorpyrifos were performed. Several endpoints, such as swimming behaviour, morphological abnormalities and mortality, were studied. In addition, the inhibition of acetylcholinesterase (AChE) activity was investigated in order to evaluate the mode of action and toxicity of chlorpyrifos in the presence of these herbicides. Results indicate that both binary mixtures elicited synergistic responses on the swimming behaviour of zebrafish larvae. Moreover, although the herbicides were not effective inhibitors of the AChE on their own, a synergistic inhibition of the enzyme activity was obtained by exposure to mixtures with chlorpyrifos. We observed a correlation between impairment of swimming behaviour of the larvae and inhibition of AChE activity. This study supports previous studies concerning the risk assessment of mixtures since the toxicity may be underestimated when looking only at the single toxicants and not their mixtures.

Characterization of cholinesterases in Chironomus riparius and the effects of three herbicides on chlorpyrifos toxicity.

In this study, the toxicities of four pesticides (the herbicides atrazine, terbuthylazine, metolachlor and the insecticide chlorpyrifos) previously detected in the Alqueva reservoir/dam (south of Portugal) were evaluated individually and in binary combinations of the herbicides and the insecticide using fourth-instar larvae of the aquatic midge Chironomus riparius. Chlorpyrifos induced toxicity to midges in all the 48 h toxicity bioassays performed. The swimming behaviour of the larvae was impaired, with EC50 values ranging from 0.15 to 0.17 μg/L. However, neither s-triazine (atrazine and terbuthylazine) herbicides nor metolachlor alone at concentrations up to 200 μg/L caused significant toxicity to C. riparius. When combined with both s-triazine herbicides, chlorpyrifos toxicity was enhanced by approximately 2-fold when tested in a binary mixture experimental setup, at the 50% effective concentration levels. To evaluate how chlorpyrifos toxicity was being increased, the cholinesterases (ChE) were characterized biochemically using different substrates and selective inhibitors. The results obtained suggested that the main enzyme present in this species is acetylcholinesterase (AChE) and therefore it was assayed upon C. riparius exposures to all pesticides individually and as binary mixtures. Although atrazine and terbuthylazine are not effective inhibitors of AChE, the potentiation of chlorpyrifos toxicity by the two s-triazine herbicides was associated with a potentiation in the inhibition of AChE in midges; both s-triazine herbicides at 200 μg/L increased the inhibition of the AChE activity by 7 and 8-fold, respectively. A strong correlation was observed between swimming behaviour disturbances of larvae and the inhibition of the AChE activity. In contrast, metolachlor did not affect chlorpyrifos toxicity at any of the concentrations tested. Therefore, the herbicides atrazine and terbuthylazine can act as synergists in the presence of chlorpyrifos, increasing the toxicity and consequently underestimating risk based on single chemical levels.

Does subcellular distribution in plants dictate the trophic bioavailability of cadmium to Porcellio dilatatus (crustacea, isopoda)?

The present study examined how subcellular partitioning of Cd in plants with different strategies to store and detoxify Cd may affect trophic transfer of Cd to the isopod Porcellio dilatatus. The plant species used were Lactuca sativa, a horticultural metal accumulator species; Thlaspi caerulescens, a herbaceous hyperaccumulator species; and the nonaccumulator, T. arvense. Taking into account that differences in subcellular distribution of Cd in plants might have an important role in the bioavailability of Cd to a consumer, a differential centrifugation technique was adopted to separate plant leaf tissues into four different fractions: cell debris, organelles, heat-denatured proteins, and heat-stable proteins (metallothionein-like proteins). Plants were grown in replicate hydroponic systems and were exposed for 7 d to 100 microM Cd spiked with 109Cd. After a 14-d feeding trial, net assimilation of Cd in isopods following consumption of T. caerulescens and T. arvense leaves reached 16.0 +/- 2.33 and 21.9 +/- 1.94 microg/g animal, respectively. Cadmium assimilation efficiencies were significantly lower in isopods fed T. caerulescens (10.0 +/- 0.92%) than in those fed T. arvense (15.0 +/- 1.03%). In further experiments, Cd assimilation efficiencies were determined among isopods provided with purified subcellular fractions of the three plants. On the basis of our results, Cd bound to heat-stable proteins was the least bioavailable to isopods (14.4-19.6%), while Cd bound to heat-denatured proteins was the most trophically available to isopods (34.4-52.8%). Assimilation efficiencies were comparable in isopods fed purified subcellular fractions from different plants, further indicating the importance of subcellular Cd distribution in the assimilation. These results point to the ecological relevance of the subcellular Cd distribution in plants, which directly influence the trophic transfer of Cd to the animal consumer.

Endocrine disruption effects of p,p'-DDE on juvenile zebrafish.

The persistent organic pollutant p,p′‐DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p′‐DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollutant in zebrafish (Danio rerio), juveniles (30 days post hatch) were exposed to p,p′‐DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 µg l–1 over 14 days and were maintained in control water for an additional 4 months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4 months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 µg l–1 p,p′‐DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impair their successful reproduction. These results demonstrate the ability of p,p′‐DDE to cause endocrine disruption in zebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p′‐DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish. Copyright

The influence of metal speciation on the bioavailability and sub-cellular distribution of cadmium to the terrestrial isopod, Porcellio dilatatus.

Cadmium is a non-essential toxic metal that is able to bioaccumulate in both flora fauna and has the potential to biomagnify in some food chains. However, the form in which cadmium is presented to consumers can alter the bioavailability and possibly the internal distribution of assimilated Cd. Previous studies in our laboratory highlighted differences in Cd assimilation among isopods when they were provided with a plant-based food with either Cd biologically incorporated into plant tissue or superficially amended with ionic Cd(2+). Cd is known for its high affinity for sulphur ligands in cysteine residues which form the basis for metal-binding proteins such as metallothionein. This study compares Cd assimilation efficiency (AE) in Porcellio dilatatus fed with food amended with either cadmium cysteinate or cadmium nitrate in an examination of the influence of Cd speciation on metal bioavailability followed by an examination of the sub-cellular distribution using a centrifugal fractionation protocol. As hypothesized the AE of Cd among isopods fed with Cd(NO(3))(2) (64%, SE=5%) was higher than AE for isopods fed with Cd(Cys)(2) (20%, SE=3%). The sub-cellular distribution also depended on the Cd species provided. Those isopods fed Cd(Cys)(2) allocated significantly more Cd to the cell debris and organelles fractions at the expense of allocation to metal-rich granules (MRG). The significance of the difference in sub-cellular distribution with regard to toxicity is discussed. This paper demonstrates that the assimilation and internal detoxification of Cd is dependent on the chemical form of Cd presented to the isopod.

Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxification capacities.

Cadmium (Cd) is a widespread environmental contaminant, strongly mutagenic and known to cause DNA damage in plants. In this work, flow cytometry (FCM) was applied to determine if in vivo exposure to Cd would induce genotoxic effects at the genome level. The hyper-accumulator Thlaspi caerulescens (J. & C. Presl), the related non-accumulator Thlaspi arvense L. and the accumulator crop species Lactuca sativa L. were germinated in distilled water and grown in modified Hoaglands medium with increasing concentrations of Cd(NO3)2 (0, 1, 10 and 100 microM). After 28 days of exposure, shoot and root growth was recorded and the tissues were harvested for Cd and FCM analysis. In general, roots from treated plants contained higher content of Cd than leaves and growth inhibition was observed in the treated plants. Nuclear DNA content was estimated and the G0/G1 full peak coefficient of variation (FPCV), as an indicator of clastogenic damage, was recorded. In T. arvense and T. caerulescens no significant differences were detected between control and exposed plants. Leaves of L. sativa exposed to 10 microM Cd presented a statistically significant increase in FPCV values in comparison with the control group. Furthermore, roots exposed to 100 microM Cd presented a reduction in nuclear DNA content and an increase in FPCV when compared to the control. FCM data indicates that no major DNA damage was induced on both Cd-exposed Thlaspi species and L. sativa leaves. On the contrary, results obtained with L. sativa roots suggests clastogenic damage in these organs exposed to 100 microM of Cd.

Microsatellite instability in Lactuca sativa chronically exposed to cadmium

Cadmium (Cd) is a cytotoxic, mutagenic and carcinogenic metal. Mutagenesis is indicative of genetic instability and can be assayed by use of microsatellite markers (simple sequence repeats, SSRs). These are tandem-repeated tracts of DNA composed of units that are 1-6 base pairs (bp) long, spread throughout the genome and highly polymorphic. SSRs can be used in the detection of genomic DNA damage and/or mutational events (e.g. deletions, insertions, point mutations). In order to study chronic exposure to cadmium, Lactuca sativa L. seeds were germinated in distilled water and grown on modified Hoaglands medium, both supplemented with 0, 10 and 100microM Cd(NO(3))(2). After 28 days of exposure, the plants were harvested to assess shoot and root length and accumulation of cadmium. DNA was extracted from young and expanded leaves and roots in order to analyse microsatellite instability (MSI). Mutagenic effects of cadmium were evaluated on nine microsatellite loci. No MSI was found in leaves, but a 2-bp deletion in one lettuce root SSR was detected among the SSRs that were analysed. Thus, SSR analyses may provide a complementary tool in the assessment of different genotoxic effects of compounds on plants.

Endocrine and physiological effects of linuron and S-metolachlor in zebrafish developing embryos

Evaluation of the effects of linuron and S-metolachlor on apical, biochemical and transcriptional endpoints in zebrafish (Danio rerio) early life stages was the main purpose of this work. Embryos were exposed for 96h to a range of concentrations of each herbicide to determine lethal and sub-lethal effects on apical (e.g. malformations, hatching) and biochemical parameters (cholinesterase, ChE; catalase, CAT; glutathione S-transferase, GST; lipid peroxidation, LPO and lactate dehydrogenase, LDH). To evaluate endocrine disruption effects, embryos were exposed during 96h to 0.88mg/L linuron and 9.66mg/L S-metolachlor, isolated or in binary mixture. Expression of a suite of genes involved in HPT, HPG and HPA-axis was then assessed. Highest concentration of linuron (5.0mg/L) decreased hatching rate to 5% and 70.0mg/L S-metolachlor completely inhibited hatching, about 100%. Both herbicides impaired development by inducing several malformations (100% in 5.0mg/L linuron and 70.0mg/L S-metolachlor). Linuron only affected GST and CAT at concentrations of 0.25 and 0.0025mg/L, respectively. S-metolachlor induced GST (to 256%), inhibited ChE (to 61%) and LDH (to 60%) and reduced LPO levels (to 63%). Linuron isolated treatment seems to have an estrogenic mode of action due to the observed induction of vtg1. Exposure to S-metolachlor seems to interfere with steroidogenesis and with HPT and HPA-axis, since it has inhibited cyp19a2, TSHβ and CRH gene expression. In addition to vtg1 induction and CRH inhibition, herbicide combination also induced sox9b that has a role in regulation of sexual development in zebrafish. This study pointed out adverse effects of linuron and S-metolachlor, namely impairment of neurotransmission and energy production, induction of steroidogenesis, and interference with HPT and HPA-axis. These results contributed to elucidate modes of action of linuron and S-metolachlor in zebrafish embryo model. Furthermore, gene expression patterns obtained are indicative of endocrine disruption action of these herbicides.

Characterization of Cholinesterases in Plasma of Three Portuguese Native Bird Species: Application to Biomonitoring

Over the last decades the inhibition of plasma cholinesterase (ChE) activity has been widely used as a biomarker to diagnose organophosphate and carbamate exposure. Plasma ChE activity is a useful and non-invasive method to monitor bird exposure to anticholinesterase compounds; nonetheless several studies had shown that the ChE form(s) present in avian plasma may vary greatly among species. In order to support further biomonitoring studies and provide reference data for wildlife risk-assessment, plasma cholinesterase of the northern gannet (Morus bassanus), the white stork (Ciconia ciconia) and the grey heron (Ardea cinerea) were characterized using three substrates (acetylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulphate, BW284C51, and iso-OMPA). Additionally, the range of ChE activity that may be considered as basal levels for non-exposed individuals was determined. The results suggest that in the plasma of the three species studied the main cholinesterase form present is butyrylcholinesterase (BChE). Plasma BChE activity in non-exposed individuals was 0.48±0.11 SD U/ml, 0.39±0.12 SD U/ml, 0.15±0.04 SD U/ml in the northern gannet, white stork and grey heron, respectively. These results are crucial for the further use of plasma BChE activity in these bird species as a contamination bioindicator of anti-cholinesterase agents in both wetland and marine environments. Our findings also underscore the importance of plasma ChE characterization before its use as a biomarker in biomonitoring studies with birds.