Temporal and Spatial Expression Patterns of Sox1 Gene in Xenopus laevis Embryo

We describe the temporal and spatial expression pattern of Sox 1 gene during Xenopus laevis early development and compare the expression patterns of Sox 1-3 in the developing eye and brain. Alignment of Sox 1-3 amino acid sequences shows a high conservation within the HMG-box DNA binding domains. RT-PCR analysis indicates that Sox 1 is expressed throughout development from the unfertilized egg to at least the tadpole stage, although at different expression levels. The transcripts of XSox 1 are detected in the animal pole at cleavage and blastrula stages and mainly in the central nervous system （CNS） and the developing eye at neurula stages. The study of the developmental expression of XSox 1 will aid in the elucidation of the function of SoxB 1 subgroup genes in vertebrate neurogenesis.

Effects of Nutritional Factors on the Growth and Heterotrophic Eicosapentaenoic Acid Production of Diatom Nitzschia laevis

The effects of several nutritional factors on the growth and eicosapentaenoic acid （EPA） production of diatom Nitzschia laevis were studied. 4LDM （quadrupled concentration of the nutrient salt） was the optimal concentration of nutrient salt for the growth and EPA production ofN. laevis. The growth ofN. laevis was inhibited when the glucose concentration was either lower than 10gL^-1 or higher than 15 gL^-1. Both sodium nitrate and urea were good nitrogen sources for the growth and EPA production, while ammonium chloride seriously decreased the dry cell weight （DW） and the EPA content. Silicate seriously influenced the growth of N. laevis. The maximum DW of 2.34 gL^-1 was obtained in the presence of 150 mgL^-1 Na2SiO3· 9H2O. The EPA content remained almost the same when the silicate concentration was lower than 150mgL^-1; however, higher silicate concentrations resulted in a steady decrease of EPA content. Low medium salinity （≤29） did not seem to influence the DW of N. laevis, and high salinity resulted in a decrease of DW. The highest EPA content （4.08%） and yield （110mgL^-1） were observed at the salinity of 36 and 29, respectively. Key words polyunsaturated fatty acid; eicosapentaenoic acid; microalga; Nitzschia laevis; heterotrophy

Thyroid disruption by technical decabromodiphenyl ether (DE-83R) at low concentrations in Xenopus laevis

Decabromodiphenyl ether （decaBDE）,as a flame retardant,is widely produced and used.To study the thyroid disruption by technical decaBDE at low concentrations,Xenopus laevis tadpoles were exposed to technical decaBDE mixture DE-83R （1-1000 ng/L） in water from stage 46/47 （free swimming larvae,system of Nieuwkoop and Faber） to stage 62.DE-83R at concentration of 1000 ng/L significantly delayed the time to metamorphosis （presented by forelimb emergence,FLE）.Histological examination showed that DE83R at all tested concentrations caused histological alterations-multilayer follicular epithelial cell and markedly increased follicle size accompanied by partial colloid depletion and increase in the peripheral colloid vacuolation,in thyroid glands.All tested concentrations of DE-83R also induced a down-regulation of thyroid receptor mRNA expression.These results demonstrated that technical decaBDE disrupted the thyroid system in X.laevis tadpoles.Analysis of polybrominated diphenyl ethers （PBDEs） （sum of 39 congeners） in X.laevis indicated that mean concentrations of total PBDEs in X.laevis exposed to 1,10,100,1000 ng/L were 11.0,128.1,412.1,1400.2 ng/g wet weight,respectively.Considering that PBDEs burden of X.laevis tadpoles was close to PBDEs levels in amphibians as reported in previous studies,our study has raised new concerns for thyroid disruption in amphibians of technical decaBDE at environmentally relevant concentrations.

Biochemical and Haematological Effects of the Leaf Extract of <i>Newbouldia laevis</i>in Alloxan-Induced Diabetic Rats

Dichloromethane-methanol (1:1) extract (DME) of N. laevis leaves was prepared by cold maceration. The effects of the extract on the haematological and some biochemical parameters of alloxan-induced diabetic rats were investigated. The results showed that the oral administration of the extract (250, 500, 1000 mg/kg) caused a significant (P < 0.5) and dose-dependent increase in red blood cell count (RBC) and its indices, as well as a significant (p 0.05) and dose-dependent reduction in the platelet count and the white blood cells (WBC). The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly (p 0.05) decreased. This effect was not dose related. The serum levels of total bilirubin, urea and creatinine were significantly (p 0.05) decreased. The serum total protein and total antioxidant status (TAS) significantly (p 0.05) increased dose dependently. Overall, administration of DME has significant ameliorative effect on alloxan-induced anaemia and other haematological alterations in diabetes and this may be of immense benefits in the management of diabetes and its associated haematological complications. Improved liver and kidney functions as well as improved antioxidant status are beneficial in the management of chronic diseases such as diabetes.

In silico prediction of phytoconstituents from Ehretia laevis targeting TNF-αin arthritis

Objective Rheumatoid arthritis(RA)is an autoimmune disease involving the synovial lining of the major joints.Current therapies have noteworthy side effects.Our study involved in silico evaluation of Ehretia laevis(E.laevis)phytoconstituents targeting tumor necrosis factor-α(TNF-α).Methods Molecular docking studies performed to investigate the binding pattern of the plant E.laevis phytoconstituents along with the crystal structure of TNF-α(PDB ID:2 AZ5)using AutoDock Vina followed by a study of interacting amino acid residues and their influence on the inhibitory potentials of the active constituents.Further the pharmacokinetic profile and toxicity screening carried out using Swiss ADME and pk CSM.Results The docked results suggest that lupeol(-9.4 kcal/mol)andα-amyrin(-9.4 kcal/mol)has best affinity towards TNF-αcompared to standard drug thalidomide(-7.4 kcal/mol).The active chemical constituents represents better interaction with the conserved catalytic residues,leading to the inhibition/blockade of the TNF-α-associated signaling pathway in RA.Furthermore,pharmacokinetics and toxicity parameters of these phytochemicals were within acceptable limits according to ADMET studies.Conclusion The binding potential of phytoconstituents targeting TNF-αshowed promising results.Nonetheless,it encourages the traditional use of E.laevis and provides vital information on drug development and clinical treatment.

Preservative Effect of Newbouldia laevis (Boundary Tree) Leaf Extract on Shelf-Life of Fresh Chicken Meat under Tropical Conditions

The shelf life of chicken meat has been rapidly reduced as a result of high environmental temperatures prevalent in the tropics which favored the activities of spoilage micro-organisms,and reactive oxygen species that function in oxidative damage.Newbouldia laevis(N.laevis)possess valuable antioxidant and antibacterial properties.However,information on the effect of aqueous extracts of dry and wet leaves of N.laevis on preservation of fresh chicken meat under tropical condition is scanty and thus,investigated.Broiler chicken meat(10kg weight)was obtained immediately after slaughter and were randomly allotted to three treatments(T1-control;T2-aqueous extract of wet leaves of N.laevis;and T3-aqueous extract of dry leaves of N.laevis)in a Randomized Complete Block Design.Tropical plant,such as Newbouldia laevis with relatively high resistance to heat stress,possesses viable bioactive compounds that can lower the growth of spoilage micro-organisms and activities of reactive oxygen species on fresh chicken meat under tropical conditions for 48 hours.Hence,poultry farmers in developing nations with fluctuating power supply can adopt the quick meat shelf life enhancement technique,while commercial poultry farmers across the globe can embark on product fortification using extracts of Newbouldia laevis.

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.

Effect of antikeratin microinjection on the embryonic development of Xenopus laevis

Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.

Bioconcentration kinetics of PCBs in various parts of the lifecycle of the tadpoles Xenopus laevis

Polychlorinated biphenyls （PCBs） in Xenopus laevis have been reported only for a few congeners. Additionally, there is very little information on the ability of Xenopus laevis to bioconcentrate PCBs. To address these issues, the tadpole Xenopus laevis was exposed to Aroclor 1254 mixtures in water at room temperature for 110 d followed by an additional 110 d of nonspiked PCBs in the water for the control group. During the whole process, bioconcentration factors （BCFs） of PCBs ranged from 1180 to 15670. For most PCB congeners, the highest and lowest bioconcentrations of the kinetic curves were found to be remarkably simultaneous, respectively. All 141 PCB congeners under the same experimental conditions had no linear correlation on the lgBCF versus lgKow relationship. The relationship between lgBCFs and lgKow followed a parabolic pattern indicative of selective bioconcentration, suggesting that the kinetic curves of the PCB congeners observed in the lifecycle of the tadpoles may be concentrated due to the amphibian special species and internal metabolism. In contrast, lgBCFs for PCBs were inversely related to lgKow, suggesting that a metabolism of the higher Kow PCB congeners occurred. These results support the author＇s conclusion that the tadpole Xenopus laevis plays major roles in the bioconcentration of PCB congeners, and demonstrated that the exposure kinetic curves of PCB congeners are complex. Besides the amphibian metamorphous development, the lifecycle of the tadpole Xenopus laevis also may be of importance in determining the bioconcentration of PCB congeners.

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell-free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation, nuclear envelope assembly, and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle. The assembled nuclei, being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii. However, incubation of dinoflagellate Crythecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinillm cohnii cells does not induce nuclear reconstitution.

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.

IRE1α knockdown rescues tunicamycin-induced developmental defects and apoptosis in Xenopus laevis

Inositol requiring enzyme-1 （IRE1） is highly conserved from yeasts to humans. Upon endoplasmic reticulum （ER） stress, IRE1 activates X-box-binding protein 1 （XBP1） by unconventional splicing of XBP1 mRNA, which activates unfolded protein response （UPR） to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α （xIRE1α）. The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptofic cells were measured by TUNNEL assays. Although both gain and loss of xlRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.

A developmental biological study of aldolase gene expression in Xenopus laevis

We cloned cDNAs for Xenopus aldolases A, B and C. These three aldolase genes are localized on different chromosomes as a single copy gene. In the adult, the aldolase A gene is expressed extensively in muscle tissues, whereas the aldolase B gene is expressed strongly in kidney, liver, stomach and intestine, while the aldolase C gene is expressed in brain, heart and ovary. In oocytes aldolase A and C mRNAs, but not aldolase B mRNA, are extensively transcribed. Thus, aldolase A and C mRNAs, but not B mRNA, occur abundantly in eggs as maternal mRNAs, and strong expression of aldolase B mRNA is seen only after the late neurula stage. We conclude that aldolase A and C mRNAs are major aldolase mRNAs in early stages of Xenopus embryogenesis which proceeds utilizing yolk as the only energy source, aldolase B mRNA, on the other hand, is expressed only later in development in tissues which are required for dietary fructose metabolism.We also isolated the Xenopus aldolase C genomic gene (ca. 12 kb) and found that its promoter (ca. 2 kb)contains regions necessary for tissue-specific expression and also a GC rich region which is essential for basal transcriptional activity.

Developmental dependence for functional eye regrowth in Xenopus laevis

Introduction：A key challenge in designing tissue repair strategies is knowing whether and how developmental mechanisms are used for successful repair of mature/adult tissues.Although it is known that developmental components are used in repair,it remains mostly unclear which ones are required and whether they act similarly as during development.This issue is further complicated by the fact that it is difficult.

Antihyperglycemic Studies on the Leaf Extract and Active Fractions of <i>Newbouldia laevis</i>(Bignoniaceae)

Optimal control of chronic hyperglycemia prevents both micro and macro vascular complications—a leading cause of morbidity and mortality in diabetic subjects. This study was undertaken to give credence to the traditional use of Newbouldia laevis leaves in the treatment of diabetes mellitus (DM). Dichloromethane-methanol (1:1) extract (DME) of N. laevis leaves was prepared by cold maceration. Separation of DME into column chromatographic fractions yielded the n-hexane fraction (HF), ethylacetate fraction (EF) and methanol fraction (MF). The extract and fractions were evaluated for antihyperglycemic activity in alloxanized diabetic rats. The results showed that the oral administration of extract and fractions (250, 500, 1000 mg/kg) caused a significant (P < 0.5) and dose-dependent reduction in blood glucose level in diabetic rats. The hypoglycemic potency after 24 h was in the order MF (methanol fraction;56.31%) > DME (dichloromethane/methanol extract;36.19%) > EF (ethylacetate fraction;20.70%) > HF (n-hexane fraction;10.09. The methanol fraction, which showed the highest potency in oral glucose tolerance test (OGTT), was further separated into column chromatographic sub-fractions—F1, F2, F3 and F4 fractions. These sub-fractions were evaluated for antihyperglycemic activity. Sub-fractions F1, F2 and F3 (1000 mg/kg) did produce significant (P > 0.05) reduction in blood glucose level after 24 h. Sub-fraction F4 (50, 100, 200 mg/kg), however caused a significant (P < 0.05) and dose-dependent reduction in blood glucose level. The reduction at 200 mg/kg dose of F4 (74.57%) was significantly (P < 0.05) higher than that of glibenclamide (58.04%). These findings suggest that leaf extract and fractions of Newbouldia laevis possess antihyperglycemic activities and can be the basis for the folk use N. laevis in management of diabetes mellitus.

Expression Patterns of CAPN1 and CAPN8b Genes during Embryogenesis in Xenopus laevis

Calpains are a superfamily of Ca2+-dependent cysteine proteases, implicated in various cellular processes and thus probably necessary in all the stages of cell life. The first extended report of quantification of total RNAs within the developmental stages of Xenopus laevis was described in this study. Decreases of total RNAs were positively associated with waves of apoptotic cell death (onset of gastrulation, and morphogenesis). Using qPCR, the temporal expression pattern of CAPN1 and CPAN8b (XCL-2) were characterized during the Xenopus laevis embryogenesis. Transcripts of the CAPN1 and CAPN8 genes were detectable from gastrula stage and their levels oscillated throughout development. The expression of the CAPN1 (mu/I) gene was observed in earliest stage, indicating a maternal origin, while expression of the CAPN8b gene was detectable after midblastula transition. The levels of the two transcripts then started to rise again obviously as a result of zygotic expression (stage 11). The CAPN1 gene expression was particularly expressed at tailbud stage, while the CAPN8 transcripts were found at gastrula, neurula and tailbud stages. This is the first report of quantification of mRNAs CAPN8b and CAPN1 (mu/I) within the developmental stages of Xenopus laevis by qPCR.

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i>embryogenesis

To study gene control mechanisms in Xenopus embryos, we analyzed polyamines, cloned SAMDC (S-adenosylmethionine decarboxylase), a key enzyme of polyamine metabolism, and microinjected its mRNA into Xenopus fertilized eggs. The microinjection induced a large increase in SAMDC activity, exhaustion of the substrate SAM (S-adenosylmethionine), and execution of apoptosis at the stage called midblastula transition (MBT). By tracing GFP (green fluorescence protein)-marked apoptotic cells, we reached a conclusion that the apoptosis provides pre-blastula embryos with a fail-safe mechanism of early development. We analyzed caspase mRNAs and found that caspase-9 and -3 mRNAs are maternal mRNA and activation of caspase-9 is one of the key steps for the execution of the apoptosis. We also found that over- expression of caspase-8, and in addition p53, a tumor suppressor protein, also induces apoptosis at MBT, just like the overexpression of SAMDC and caspase-9 does. The apoptosis induced by p53 was suppressed by Xdm-2, a negative regulator of p53, and by a peptide inhibitor and a dominant-negative type mutant of caspase-9, but not by those of caspase-8. By contrast, apoptosis induced by SAMDC was suppressed by peptide inhibitors and dominant-negative mutants of both caspase-9 and caspase-8, but not by Xdm-2. Unlike caspase-9 mRNA, caspase-8 mRNA was not a maternal mRNA, but newly expressed during cleavage stage (pre-MBT stage) only in embryos overexpressed with SAMDC. In SAMDC-induced apoptotic embryos activities to process procaspase-8 and procaspase-9 appeared, whereas in p53-induced apoptotic embryos only activity to process procaspase-9 appeared. Thus, Xenopus embryos have at least two pathways to execute the maternal program of apoptosis: One induced by SAMDC overexpression through activation of caspase-9 and do novo expression of caspase-8 gene, and the other induced by p53 overexpression through activation of caspase-9 but not caspase-8. In Xenopus embryos, it has long been believed that zygotic genes are silent until MBT, but results obtained with caspase-8 may provide a novel example of gene expression before MBT.

Harmful Effects on African Clawed Frog （Xenopus laevis） Reproduction as Expression of High Water Phosphates Levels

Amphibians are considered as reliable indicators of environmental quality. In Europe, a general decline of amphibians population parallels a worldwide decline, and some of the factors which are thought to be responsible to this decline are habitat loss, introduction of exotic species, disease and decreasing water quality. Recent investigations suggest that these eutrophic conditions may be associated with frog reproduction problems. Water quality criteria settled for the different species of amphibians do not currently exist, but in the present paper, the authors report their observation about water phosphate concentrations in association with hipofertility in African clawed frogs （Xenopus laevis）. The study considered two different groups of African clawed frog showing hypofertility （22 patients） hosted in captivity. The animals were visited, and the filtration systems functionality and the water quality were checked, i.e., temperature, pH, ammonia, nitrite, nitrate, phosphates, hardness and alkalinity. In the first group, it was decided to perform ultrasound scans, parasitological tests on fecal samples, and microscopical/macroscopical examination, as well as microbiological analysis on collected oocytes. In the second group, only fecal samples were collected in order to perform parasitologiacal exams. No pathological findings were showed by veterinary tests. Water changes were carried out in both facilities and after two months, the fertility in African clawed frogs improved, evidencing the involvement of phosphates values in the onset of the problem and even more in its resolution. More studies are needed to further define this correlation.

Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunction in Adult Wistar Rats Running Title:Newbouldia laevis Ameliorates Ovarian Disruption

Background:The extract of Newbouldia laevis has been demonstrated to show antioxidant,antimicrobial,sedative,anticonvulsant,analgesic,anti-inflammatory,antinociceptive,hepatoprotective,anticancer,antiulcer,antihypertensive and antidiarrhea properties.However,its reproprotective effect is not well known.The present study was designed to investigate the ameliorative effect of Newbouldia laevis in cadmium-induced ovarian dysfunction.Method:Adult female Wistar rats were randomly allotted into groups;Vehicle(received distilled water),cadmium-treated(received 5 mg/kg b.w.),N.laevis-treated(received 200 mg/kg b.w.)and cadmium+N.laevis-treated groups.Cadmium sulphate was administered for 3 days(i.p.)followed by oral administration of N.laevis for 28 days.The body weight change was monitored using animal weighing balance(Olympia SCL66110 model,Kent Scientific Corporation,Torrington,CT06790,USA),biochemical assay and histology of ovaries were performed as previously described.Results:The results showed weight loss,severe disruption of ovarian follicles and significant reduction of gonadotropic hormones(FSH and LH)in cadmium-treated group compared with vehicle-treated group.These alterations were not associated with inflammatory response.However,concomitant administration of aqueous extract of N.laevis and cadmium sulphate significantly ameliorated ovarian disruption.Conclusion:The study demonstrates that administration of aqueous extract of N.laevis during treatment with cadmium sulphate preserves ovarian function,suggesting that possibly daily intake of aqueous extract of N.laevis prevents the onset of ovarian disorders.

Low concentrations of atrazine,glyphosate,2,4-dichlorophenoxyacetic acid,and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis

Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides （atrazine, 2,4-dichloropheoxyacetic acid （2,4-D）, triadimefon, and glyphosate） in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.