A bumble bee forages on a blueberry flower (left). Beekeepers inspect their colonies during almon... more A bumble bee forages on a blueberry flower (left). Beekeepers inspect their colonies during almond pollination (right).
Leaves were sprayed with dinotefuran or Safari and left outside to be exposed to the elements. Pe... more Leaves were sprayed with dinotefuran or Safari and left outside to be exposed to the elements. Periodically, a set of leaves were picked and honey bees were exposed for 24 hours. To extract dinotefuran or Safari on the exterior of bees in preparation for analysis, 5 bees were placed in 5 mL of distilled water. 1 μg/mL of d3-dinotefuran (Toronto Research Chemicals, Toronto, Canada) was included as an internal standard. The bees were vortexed using a Multi-Tube Holder attached to a Vortex Genie 2 (Scientific Industries, Bohema, NY) for 4 minutes at maximum power. 1 mL of the water was transferred to a 1.5 mL plastic tube, which was vortexed at 5,000 rcf for 5 minutes. 100 μL of the supernatant was transferred to a 300 μL polypropylene autosampler vial (MicroSolv Technology Corp., Eatontown, NJ).
The decline in populations of insect pollinators is a global concern. While multiple factors are ... more The decline in populations of insect pollinators is a global concern. While multiple factors are implicated, there is uncertainty surrounding the contribution of certain groups of pesticides to losses in wild and managed bees. Nanotechnology-based pesticides (NBPs) are formulations based on multiple particle sizes and types. By packaging active ingredients in engineered particles, NBPs offer many benefits and novel functions, but may also exhibit different properties in the environment when compared with older pesticide formulations. These new properties raise questions about the environmental disposition and fate of NBPs and their exposure to pollinators. Pollinators such as honey bees have evolved structural adaptations to collect pollen, but also inadvertently gather other types of environmental particles which may accumulate in hive materials. Knowledge of the interaction between pollinators, NBPs, and other types of particles is needed to better understand their exposure to pes...
The decline in populations of insect pollinators is a global concern. While multiple factors are ... more The decline in populations of insect pollinators is a global concern. While multiple factors are implicated, there is uncertainty surrounding the contribution of certain groups of pesticides to losses in wild and managed bees. Nanotechnology-based pesticides (NBPs) are formulations based on multiple particle sizes and types. By packaging active ingredients in engineered particles, NBPs offer many benefits and novel functions, but may also exhibit different properties in the environment when compared with older pesticide formulations. These new properties raise questions about the environmental disposition and fate of NBPs and their exposure to pollinators. Pollinators such as honey bees have evolved structural adaptations to collect pollen, but also inadvertently gather other types of environmental particles which may accumulate in hive materials. Knowledge of the interaction between pollinators, NBPs, and other types of particles is needed to better understand their exposure to pes...
Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which... more Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.
ABSTRACT Background/Question/Methods The proportion of female PhD recipients entering science, te... more ABSTRACT Background/Question/Methods The proportion of female PhD recipients entering science, technology, engineering and mathematics (STEM) fields has increased over the past decade, but attrition exists at each promotion step. Research demonstrates that the low proportion of female professors is due in part to a reduced number of female applicants, not discrimination against women in the hiring and promotion process. Women choose to remove themselves from the academic trajectory. Efforts reduce this attrition have typically focused on laboratory sciences, and there have been few initiatives for women in disciplines such as field biology, ecology and earth systems sciences (BEESS). Scientists in BEESS fields face unique challenges: fieldwork requires managing logistics; interfacing with diverse crews and stakeholders; negotiating time away from personal and professional relationships; and re-entering into relationships upon one’s return. For women, these challenges can be compounded by responsibilities for family interactions. To address these challenges, we developed a workshop, Advancing Toward Professorship in Biology, Ecology and Earth Systems Sciences (ATPinBEESS), to provide skills for early-career academic scientists to support fulfilling careers. Prior to the workshop, we evaluated participants’ experience and perception of success in: teaching, research, publication, managing field and lab logistics, network support, and negotiating employment terms. Results/Conclusions The ATPinBEESS workshop invited 34 participants from 16 institutions and 8 states. Pre-workshop surveys indicate that most participants lacked confidence in their ability to negotiate a salary or employment package, and to collaborate outside of their discipline. Responses suggests that sessions on fostering collaborations, networking, finding and being a good mentor, overcoming fieldwork challenges, navigating tenure, interviewing and negotiating a job offer, and maintaining work-life balance, succeeded in developing career advancement skills. Networking sessions within the workshop produced a supportive community that plans to continue to communicate about future challenges. Surveys show that the confidence to achieve a tenured position was positively correlated with the confidence to write grant proposals (r (1,22)=0.47, p<0.05) and also with participant experience doing so (r (1,22)=0.47, p<0.05). However, confidence to achieve a tenured position was negatively correlated with time spent teaching (r (1,22)=-0.69). Responses from those who aspired to R1 institutions were negatively correlated with response from those who aspired to liberal arts universities (r (1,22)=-0.56). These pre-survey results are part of ongoing work to assess the participants’ perceptions of confidence through time. Future efforts to advance careers of women in STEM fields may be improved by considering discipline-specific influences on women’s career choices.
Encapsulation of pesticide active ingredients in polymers has been widely employed 16 to control ... more Encapsulation of pesticide active ingredients in polymers has been widely employed 16 to control the release of poorly water-soluble active ingredients. Given the high dispersibility 17 of these encapsulated pesticides in water, they are expected to behave differently compared to 18 their active ingredients; however, our current understanding of the fate and effects of 19 encapsulated pesticides is still limited. In this study, we employed a central composite design 20 (CCD) to investigate how pH and ionic strength (IS) affect the hydrodynamic diameter (HDD) 21 and zeta potential of encapsulated λ-cyhalothrin and how those changes affect the exposure 22 and toxicity to Daphnia magna. R 2 values greater than 0.82 and 0.84 for HDD and zeta 23 potential, respectively, irrespective of incubation time suggest those changes could be 24 predicted as a function of pH and IS. For HDD, the linear factor of pH and quadratic factor of 25 pH×pH were found to be the most significant factors affecting the change of HDD at the 26 beginning of incubation, whereas the effects of IS and IS×IS became significant as incubation 27 time increased. For zeta potential, the linear factor of IS and quadratic factor of IS×IS were 28 found to be the most dominant factors affecting the change of zeta potential of encapsulated 29 λ-cyhalothrin, irrespective of incubation time. The toxicity tests with D. magna under 30 exposure conditions in which HDD or zeta potential of encapsulated λ-cyhalothrin was 31 maximized or minimized in the overlying water also clearly showed the worst-case exposure 32 condition to D. magna was when the encapsulated λ-cyhalothrin is either stable or small in 33 the overlying water. Our results show that water quality could modify the fate and toxicity of 34 encapsulated λ-cyhalothrin in aquatic environments, suggesting understanding their aquatic 35 interactions are critical in environmental risk assessment. Herein, we discuss the implications 36 of our findings for risk assessment.
Circadian clocks allow organisms to adapt biochemical and physiological processes to their enviro... more Circadian clocks allow organisms to adapt biochemical and physiological processes to their environment in rhythms entrained by the solar day. There is intriguing evidence that the toxicological response to a number of xenobiotic compounds varies with time of day in many species, including humans. From these reports, it can be inferred that organisms may protect themselves from environmental toxins by increasing molecular defenses when daily exposure is most anticipated. By monitoring the mortality of Drosophila melanogaster exposed to a series of doses of selected pesticides at different times of day, we have established that a daily rhythm in susceptibility to many of these pesticides occurs. Dose response curves from time points in the middle of the day and night reveal that these are significant differences. Initial assays have indicated that this rhythm is abolished when flies are exposed to constant light. Multiple microarray studies have implied that constitutive expression of...
Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle dise... more Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle disease in ruminant livestock. In this study, mice were fed a selenium-deficient diet for three generations. Female mice from the third depleted generation of these mice were given water containing either no added selenium or 0.1 or 1.0 ppm selenium as sodium selenate; DNA microarrays were used to compare gene expression in the muscle from mice fed the selenium diets to that from mice remaining on the depleted diet. The most prominent expression increases were observed with Ptger2 (a prostaglandin E receptor), Tcrb-V13 (a T-cell receptor beta), Tcf-7 (a T-cell transcription factor), and Lck (lymphocyte protein tyrosine kinase), and the major consistent decrease was Vav2, an oncogene in mice consuming the selenium containing diets.
Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which... more Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.
Daily rhythms generated by the circadian clock regulate many life functions, including responses ... more Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The e-isoform of the PAR-domain protein 1 (Pdp1e) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1e increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and a-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity.
ABSTRACT Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 ... more ABSTRACT Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in human diploid fibroblasts (HDF). We found that treatment of HDF with the diol epoxide of DB[ a,l ]P causes cell cycle arrest in G 1 . An increase in DNA adduct formation with increase in concentration of dibenzo[ a,l ]pyrene diol epoxide {( m )- anti -DB[ a,l ]PDE} was also observed.
To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 4... more To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 404 base pair truncations of genomic DNA lying immediately upstream of the SeW coding sequence were cloned into a luciferase reporter vector (pGL3-Basic from Promega, Madison, WI, USA). 3656 and 406 base pair mouse SeW promoter constructs were also compared. SeW promoter activity was assayed in two rat cell lines: L8 muscle cells and C6 brain cells. The SeW promoter was 2-7 times more active (p < 0.01) than SV40 promoter. Promoter activity of constructs of the SeW promoter ranging from 200 base pairs to 51 base pairs gradually decreased to zero in brain cells, but fell precipitously to zero in muscle cells. Some truncations stimulated promoter activity, suggesting the full-length promoter may contain binding sites for factors that suppress SeW expression.
In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanili... more In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 lM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 lM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.
Background: Circadian clocks govern daily physiological and molecular rhythms, and putative rhyth... more Background: Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in expression of xenobiotic metabolizing (XM) genes have been described in both insects and mammals. Such rhythms could have important consequences for outcomes of chemical exposures at different times of day. To determine whether reported XM gene expression rhythms result in functional rhythms, we examined daily profiles of enzyme activity and dose responses to the pesticides propoxur, deltamethrin, fipronil, and malathion.
A bumble bee forages on a blueberry flower (left). Beekeepers inspect their colonies during almon... more A bumble bee forages on a blueberry flower (left). Beekeepers inspect their colonies during almond pollination (right).
Leaves were sprayed with dinotefuran or Safari and left outside to be exposed to the elements. Pe... more Leaves were sprayed with dinotefuran or Safari and left outside to be exposed to the elements. Periodically, a set of leaves were picked and honey bees were exposed for 24 hours. To extract dinotefuran or Safari on the exterior of bees in preparation for analysis, 5 bees were placed in 5 mL of distilled water. 1 μg/mL of d3-dinotefuran (Toronto Research Chemicals, Toronto, Canada) was included as an internal standard. The bees were vortexed using a Multi-Tube Holder attached to a Vortex Genie 2 (Scientific Industries, Bohema, NY) for 4 minutes at maximum power. 1 mL of the water was transferred to a 1.5 mL plastic tube, which was vortexed at 5,000 rcf for 5 minutes. 100 μL of the supernatant was transferred to a 300 μL polypropylene autosampler vial (MicroSolv Technology Corp., Eatontown, NJ).
The decline in populations of insect pollinators is a global concern. While multiple factors are ... more The decline in populations of insect pollinators is a global concern. While multiple factors are implicated, there is uncertainty surrounding the contribution of certain groups of pesticides to losses in wild and managed bees. Nanotechnology-based pesticides (NBPs) are formulations based on multiple particle sizes and types. By packaging active ingredients in engineered particles, NBPs offer many benefits and novel functions, but may also exhibit different properties in the environment when compared with older pesticide formulations. These new properties raise questions about the environmental disposition and fate of NBPs and their exposure to pollinators. Pollinators such as honey bees have evolved structural adaptations to collect pollen, but also inadvertently gather other types of environmental particles which may accumulate in hive materials. Knowledge of the interaction between pollinators, NBPs, and other types of particles is needed to better understand their exposure to pes...
The decline in populations of insect pollinators is a global concern. While multiple factors are ... more The decline in populations of insect pollinators is a global concern. While multiple factors are implicated, there is uncertainty surrounding the contribution of certain groups of pesticides to losses in wild and managed bees. Nanotechnology-based pesticides (NBPs) are formulations based on multiple particle sizes and types. By packaging active ingredients in engineered particles, NBPs offer many benefits and novel functions, but may also exhibit different properties in the environment when compared with older pesticide formulations. These new properties raise questions about the environmental disposition and fate of NBPs and their exposure to pollinators. Pollinators such as honey bees have evolved structural adaptations to collect pollen, but also inadvertently gather other types of environmental particles which may accumulate in hive materials. Knowledge of the interaction between pollinators, NBPs, and other types of particles is needed to better understand their exposure to pes...
Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which... more Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.
ABSTRACT Background/Question/Methods The proportion of female PhD recipients entering science, te... more ABSTRACT Background/Question/Methods The proportion of female PhD recipients entering science, technology, engineering and mathematics (STEM) fields has increased over the past decade, but attrition exists at each promotion step. Research demonstrates that the low proportion of female professors is due in part to a reduced number of female applicants, not discrimination against women in the hiring and promotion process. Women choose to remove themselves from the academic trajectory. Efforts reduce this attrition have typically focused on laboratory sciences, and there have been few initiatives for women in disciplines such as field biology, ecology and earth systems sciences (BEESS). Scientists in BEESS fields face unique challenges: fieldwork requires managing logistics; interfacing with diverse crews and stakeholders; negotiating time away from personal and professional relationships; and re-entering into relationships upon one’s return. For women, these challenges can be compounded by responsibilities for family interactions. To address these challenges, we developed a workshop, Advancing Toward Professorship in Biology, Ecology and Earth Systems Sciences (ATPinBEESS), to provide skills for early-career academic scientists to support fulfilling careers. Prior to the workshop, we evaluated participants’ experience and perception of success in: teaching, research, publication, managing field and lab logistics, network support, and negotiating employment terms. Results/Conclusions The ATPinBEESS workshop invited 34 participants from 16 institutions and 8 states. Pre-workshop surveys indicate that most participants lacked confidence in their ability to negotiate a salary or employment package, and to collaborate outside of their discipline. Responses suggests that sessions on fostering collaborations, networking, finding and being a good mentor, overcoming fieldwork challenges, navigating tenure, interviewing and negotiating a job offer, and maintaining work-life balance, succeeded in developing career advancement skills. Networking sessions within the workshop produced a supportive community that plans to continue to communicate about future challenges. Surveys show that the confidence to achieve a tenured position was positively correlated with the confidence to write grant proposals (r (1,22)=0.47, p&lt;0.05) and also with participant experience doing so (r (1,22)=0.47, p&lt;0.05). However, confidence to achieve a tenured position was negatively correlated with time spent teaching (r (1,22)=-0.69). Responses from those who aspired to R1 institutions were negatively correlated with response from those who aspired to liberal arts universities (r (1,22)=-0.56). These pre-survey results are part of ongoing work to assess the participants’ perceptions of confidence through time. Future efforts to advance careers of women in STEM fields may be improved by considering discipline-specific influences on women’s career choices.
Encapsulation of pesticide active ingredients in polymers has been widely employed 16 to control ... more Encapsulation of pesticide active ingredients in polymers has been widely employed 16 to control the release of poorly water-soluble active ingredients. Given the high dispersibility 17 of these encapsulated pesticides in water, they are expected to behave differently compared to 18 their active ingredients; however, our current understanding of the fate and effects of 19 encapsulated pesticides is still limited. In this study, we employed a central composite design 20 (CCD) to investigate how pH and ionic strength (IS) affect the hydrodynamic diameter (HDD) 21 and zeta potential of encapsulated λ-cyhalothrin and how those changes affect the exposure 22 and toxicity to Daphnia magna. R 2 values greater than 0.82 and 0.84 for HDD and zeta 23 potential, respectively, irrespective of incubation time suggest those changes could be 24 predicted as a function of pH and IS. For HDD, the linear factor of pH and quadratic factor of 25 pH×pH were found to be the most significant factors affecting the change of HDD at the 26 beginning of incubation, whereas the effects of IS and IS×IS became significant as incubation 27 time increased. For zeta potential, the linear factor of IS and quadratic factor of IS×IS were 28 found to be the most dominant factors affecting the change of zeta potential of encapsulated 29 λ-cyhalothrin, irrespective of incubation time. The toxicity tests with D. magna under 30 exposure conditions in which HDD or zeta potential of encapsulated λ-cyhalothrin was 31 maximized or minimized in the overlying water also clearly showed the worst-case exposure 32 condition to D. magna was when the encapsulated λ-cyhalothrin is either stable or small in 33 the overlying water. Our results show that water quality could modify the fate and toxicity of 34 encapsulated λ-cyhalothrin in aquatic environments, suggesting understanding their aquatic 35 interactions are critical in environmental risk assessment. Herein, we discuss the implications 36 of our findings for risk assessment.
Circadian clocks allow organisms to adapt biochemical and physiological processes to their enviro... more Circadian clocks allow organisms to adapt biochemical and physiological processes to their environment in rhythms entrained by the solar day. There is intriguing evidence that the toxicological response to a number of xenobiotic compounds varies with time of day in many species, including humans. From these reports, it can be inferred that organisms may protect themselves from environmental toxins by increasing molecular defenses when daily exposure is most anticipated. By monitoring the mortality of Drosophila melanogaster exposed to a series of doses of selected pesticides at different times of day, we have established that a daily rhythm in susceptibility to many of these pesticides occurs. Dose response curves from time points in the middle of the day and night reveal that these are significant differences. Initial assays have indicated that this rhythm is abolished when flies are exposed to constant light. Multiple microarray studies have implied that constitutive expression of...
Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle dise... more Nutritional selenium deficiency is associated with Keshan disease in humans and white muscle disease in ruminant livestock. In this study, mice were fed a selenium-deficient diet for three generations. Female mice from the third depleted generation of these mice were given water containing either no added selenium or 0.1 or 1.0 ppm selenium as sodium selenate; DNA microarrays were used to compare gene expression in the muscle from mice fed the selenium diets to that from mice remaining on the depleted diet. The most prominent expression increases were observed with Ptger2 (a prostaglandin E receptor), Tcrb-V13 (a T-cell receptor beta), Tcf-7 (a T-cell transcription factor), and Lck (lymphocyte protein tyrosine kinase), and the major consistent decrease was Vav2, an oncogene in mice consuming the selenium containing diets.
Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which... more Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.
Daily rhythms generated by the circadian clock regulate many life functions, including responses ... more Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The e-isoform of the PAR-domain protein 1 (Pdp1e) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1e increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and a-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity.
ABSTRACT Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 ... more ABSTRACT Benzo[ a ]pyrene (B[ a ]P) and dibenzo[ a,l ]pyrene (DB[ a,l ]P) induce cytochrome P450 (CYP) CYP1A1 and CYP1B1, which metabolize these polycyclic aromatic hydrocarbons (PAHs) into DNA-binding species. In order to detail roles of CYP1A1 and CYP1B1 in activation of DB[ a,l ]P to the diol epoxide, we here report the inhibition of CYP1A1 in human MCF-7 cells with phosphorodiamidate morpholino antisense oligomers (morpholinos). PAH-DNA adduct formation was also determined after treatment with morpholinos and B[ a ]P or DB[ a,l ]P. p53 is involved in DNA repair, cell cycle arrest, and apoptosis. Cells with normal p53 protein arrest in the G1 phase of the cell cycle on exposure to DNA-damaging agents (presumably allowing the cell sufficient time to repair damaged DNA prior to replication). Previous studies in human MCF-7 cells indicate that cells with PAH-DNA adducts escape cell cycle arrest and accumulate in the S phase. In the present study the effect of PAH-DNA adducts on the cell cycle were observed in human diploid fibroblasts (HDF). We found that treatment of HDF with the diol epoxide of DB[ a,l ]P causes cell cycle arrest in G 1 . An increase in DNA adduct formation with increase in concentration of dibenzo[ a,l ]pyrene diol epoxide {( m )- anti -DB[ a,l ]PDE} was also observed.
To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 4... more To identify regulatory elements in the rat selenoprotein W (SeW) promoter, 2090, 1265, 741, and 404 base pair truncations of genomic DNA lying immediately upstream of the SeW coding sequence were cloned into a luciferase reporter vector (pGL3-Basic from Promega, Madison, WI, USA). 3656 and 406 base pair mouse SeW promoter constructs were also compared. SeW promoter activity was assayed in two rat cell lines: L8 muscle cells and C6 brain cells. The SeW promoter was 2-7 times more active (p < 0.01) than SV40 promoter. Promoter activity of constructs of the SeW promoter ranging from 200 base pairs to 51 base pairs gradually decreased to zero in brain cells, but fell precipitously to zero in muscle cells. Some truncations stimulated promoter activity, suggesting the full-length promoter may contain binding sites for factors that suppress SeW expression.
In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanili... more In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 lM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 lM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.
Background: Circadian clocks govern daily physiological and molecular rhythms, and putative rhyth... more Background: Circadian clocks govern daily physiological and molecular rhythms, and putative rhythms in expression of xenobiotic metabolizing (XM) genes have been described in both insects and mammals. Such rhythms could have important consequences for outcomes of chemical exposures at different times of day. To determine whether reported XM gene expression rhythms result in functional rhythms, we examined daily profiles of enzyme activity and dose responses to the pesticides propoxur, deltamethrin, fipronil, and malathion.
Uploads
Papers by Louisa Hooven