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An evaluation of acute toxicity using the phototactic behavior of Daphnia magna
e50
Abstracts
172 Age-related hearing loss in the C57BL/6N mouse: Time course analysis of auditory function with implications for auditory safety study design Matthew Abernathya,b, Rachel Tappa, Joshua Yodera, Andrea Kocha, Carolyn Hedkea, David Gauvina, Theodore Bairda a
MPI Research, Mattawan, MI, USA Western Michigan University, Kalamazoo, MI, USA
An experiment was conducted in guinea pigs in which perilymph samples were collected from three locations along the cochlea, 1) round window, 2) 4 mm from the round window, and 3) apex. The results of this experiment show that larger sample volumes can consistently be extracted from the cochlea when taken 4 mm from the round window. If applied correctly, the information from his experiment can enhance the best practices associated with perilymph collections for auditory safety evaluations.
b
Age-related hearing loss occurs in almost every species, although the time of onset and degree of loss varies from species to species. For instance, guinea pigs generally present with age-related hearing loss at 12 months of age where as some strains of mice experience hearing loss beginning at 6 weeks of age. If a species displaying agerelated hearing loss is used on an auditory safety study, it is important to understand the time course so that the study design can be modified to avoid issues with data interpretation while still assessing safety. An experiment was conducted to establish the time course of age-related hearing loss in the C57BL/6N mouse. This strain was selected as it is commonly used in auditory research and has been shown to develop auditory function deficits early in life. Beginning at 6 weeks of age, auditory brainstem response evaluations were conducted monthly until 21 weeks of age. The cochleae of 6 and 21 week old mice were processed and sectioned for hair cell quantification and histomorphological analysis on other relevant cochlear structures affecting auditory function. The results of this experiment show that the C57BL/6N strain of mouse experiences high frequency hearing loss beginning at 10 weeks of age which progresses to midrange frequencies by 21 weeks of age. Hair cell counts from 21 week old mice showed extensive loss in the basal regions of the cochlea supporting a sensorineural cause for the loss of auditory function. Options for study design modifications are discussed.
doi:10.1016/j.vascn.2013.01.178
173 Influence of collection site on cochlear perilymph sample volume Matthew Abernathya,b, Rachel Tappa, Joshua Yodera, David Gauvina, Theodore Bairda a
MPI Research, Mattawan, MI, USA Western Michigan University, Mattawan, MI, USA
b
In the context of auditory safety evaluations, it is important to assess the ability of a drug to penetrate the tympanic membrane into the middle ear. If a drug does gain access to the middle ear, it is essential to characterize exposure within the cochlea and vestibular system, the inner ear. The relationship between drug exposure and effects on hearing function must be carefully examined to determine a suitable safety margin. The perilymph of the cochlea can be utilized to evaluate inner ear exposure following intra-aural drug administration. It is well known that perilymph drug concentrations, although highly informative, can be capricious due to differences in drug distribution throughout the cochlea. Furthermore, difficulties also arise during the development of analytical methods with the capacity to detect minute concentrations of drug within microliter volume perilymph samples. These potential issues call for the identification of a cochlear site which yields larger sample volumes and, if used as a standard collection site, could overcome variances in drug distribution.
doi:10.1016/j.vascn.2013.01.179
174 Characterization of db/db mice for efficacy/safety pharmacology assessment of antidiabetic drugs Clarisse Duval, Martine Lemaire, Philippe Guillaume, Daniel Provost, Guillaume Froget Porsolt, Le Genest-Saint-Isle, France Pathological models may be relevant and powerful tools for assessing the safety of drug candidates, in parallel of their efficacy, provided they are well-characterized. Our aim was to characterize diabetic (db/db) mice, in comparison with non-diabetic lean controls in respect to their diabetic profile and associated pathological markers. Body weight and basal glycemia were higher in db/db between 7 and 15 weeks of age (34.2 ± 0.3 vs. 18.2 ± 0.2 g and 28.4 ± 1.6 vs. 11.4± 0.6 mmol/l, p b 0.001 at 8 weeks). Food and water intakes over 24 h were increased in db/db (+84% and +162%, p b 0.001). Plasma glucose and insulin were higher in db/db (33.4 ± 1.5 vs. 9.5 ± 0.9 mmol/l and 5.74 ± 0.96 vs. 0.94 ± 0.07 ng/ml, p b 0.001). After oral glucose challenge, glucose increase was relatively higher and sustained in db/db, and returned to baseline within 120 min. Insulin was under baseline from 30 min post-challenge, in contrast with controls being at baseline. Diuresis over 6 hours was higher in db/db (+0.658 g/mouse, pb 0.01). Small intestinal transit was decreased in db/db (−13%, pb 0.05). A tendency towards a decrease was observed on colonic transit. db/db mice showed a moderate hyposensitivity to tactile stimulation (Von Frey: +30%, ns; pinchmeter: +24%, pb 0.01), but a marked hypersensitivity to thermal stimulation (cold plate: −54%, p b 0.001). These findings indicate differences in db/db mice as compared with non-diabetic lean mice, in respect to their diabetic profile but also comorbidity factors. The db/db model is therefore representative of the polymorphism of diabetes, and can be a useful tool for the efficacy/safety pharmacological evaluation of antidiabetic drugs.
doi:10.1016/j.vascn.2013.01.180
175 An evaluation of acute toxicity using the phototactic behavior of Daphnia magna Justin Choi, Rosa Park, Edward Kang, Solomon Oak, Howard Yim, Mark Chung, Hye Sung Kim, Sung Hoon Choo, Jongbin Lee, Andy Suh Prafys Research Laboratory, Englewood Cliffs, NJ, USA Daphnia magna are organisms important for establishing bioassays. They serve crucial roles in pharmaceutical research to help identify chemical compounds that render adverse effects in
Abstracts
ecological environments and to screen new chemical entities for the scientific field. The purpose of this study is to provide scientists with a model that can measure the toxicity of chemicals at the earliest of stages. In this study, a test tube was placed between two light sources, located on opposite ends of an opaque plexiglass box. The illumination of the incandescent light source could be alternatively manipulated according to an experimental schedule, designated to elicit the phototactic responses of Daphnia magna. Under numerous serial dilutions of both ethyl alcohol and sodium chloride – standard chemical compounds employed by scientists – patterns in Daphnia magna phototaxis were used to examine their relationship with the acute toxicity of these compounds. The data were analyzed accordingly by using the unique phototactic index (P.I.), which calculated (a2 + b2 − c2 − d2)/100. The P.I. was plotted with respect to the concentration of the two chemical compounds, in which the slopes were defined to demonstrate the relative patterns in Daphnia phototaxis. Results of a t-test conducted statistically showed that there was a significant difference in Daphnia magna phototaxis in the two compounds, as p = 0.02791. L.J. Whitman & R.J. Miller. (1982). The Phototactic Behavior of Daphnia magna as an Indicator of Chronic Toxicity, Proc. Okla. Acad. Sci. 62, 22–33.
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177 Effect of artimisinin compound in experimentally induced colon carcinogenesis Bikash Medhi PGIMER, Chandigarh, India
Investigating an invertebrate model in safety pharmacology using the heartbeat of Daphnia magna Solomon Oak, Rosa Park, Mark Chung, Justin Choi, Edward Kang, Joseph Yang, Daniel Hong, Jamie Han, Nuri Ra, Hye Sung Kim, Sung Hoon Choo, Jongbin Lee, Andy Suh
Artemisinin, a sesquiterpene trioxane lactone had been studied for the chemopreventive efficacy in 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis in rat model. Adult Wistar rats of either sex weighing between 100 and 120 g were assorted into four groups with six animals per group. While group 1 served as control and received the vehicle of the drugs, group 2, 3 and 4 were administered weekly subcutaneous injection of 20 mg/kg DMH for 15 weeks. Group 3 was concomitantly given a weekly dose of 81 mg/kg of 5-FU intraperitoneally and the group 4 concomitantly received artemisinin orally in a dose of 82 mg/kg daily for 15 weeks. Animals were sacrificed at the end of 15 weeks, body weights recorded and the colons were subjected to macroscopic and histomorphological studies. After the experimental period, average number of ACFs/microscopic field and levels of LPO were found to be increased, whereas a significant decrease in the activities of antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione) was observed in DMHtreated rats. Artemisinin administration significantly inhibited the formation of ACFs and decreased significantly the numbers and size of tumors. In addition to this, artemisinin treatment decreased the morphological alterations of ACFs, decreased the levels of LPO and restored the activities of antioxidants to near normal levels. Furthermore artemisinin decreased the levels of pro inflammatory cytokines and induced apoptosis in the colons of DMH treated rats. Thus results obtained in the present study suggest artemisinin as an effective chemopreventive agent on colon carcinogenesis induced by DMH.
Prafys Research Laboratory, Englewood Cliffs, NJ, USA
doi:10.1016/j.vascn.2013.01.183
doi:10.1016/j.vascn.2013.01.181
176
Daphnia magna is an internationally recognized invertebrate model for various toxicological studies and bioassays. Due to the observable heartbeat changes through the transparent carapace, adverse effects of chemical compounds can be reliably detected through bioassay. However, detailed data on the daphnia with incubation duration were scanty and needed to be elaborated. The purpose of this study was to investigate the impacts on heart beat of daphnia by incubation with different duration in either environmental or pharmaceutical compounds. Twenty milliliter for an insecticide and herbicide of concentrations were serially diluted with carbonated filtered water. Six of 2 mL replications of each concentration were made in six test tubes with a micropipette. Daphnia magna were incubated in the test tubes for durations of 0.5, 1, 24, 48 and 72 hours. The heartbeat of Daphnia magna was counted before and after the incubation. For pharmaceutical compounds, dodecyl sodium sulfate and n-Dodecyl b-D-maltoside were tested with the same methods as described. The average change in heartbeat per minute was plotted on a graph with respect to the concentrations. The slope can be used to determine a concentration's effect on viability relative to other concentrations. The change in heartbeat showed a positive correlation with the concentration of the chemical compound. Through this study, establishing the adverse concentrations of these chemical compounds and possible ecological applications could be achieved. doi:10.1016/j.vascn.2013.01.182
178 Deleterious effects of gentamicin and cisplatin on renal function in rats and early detection of drug-induced kidney injury using biomarkers Eric Delpy, Marine Habault, Catherine Le Quement, Christophe Drieu La Rochelle Biotrial Pharmacology, Rennes, France Drug-induced kidney damage constitutes an important cause of acute renal failure and chronic kidney diseases in clinical setting. Unlike other organs, disturbance of kidney function is apparent only when a renal insult is well advanced. Thus, there is a need during safety pharmacology assessment to detect early injury to the kidney. The present work was carried out to evaluate renal function in conscious rats treated with gentamicin (30 and 100 mg/kg s.c. for 7 days) or cisplatin (1 and 5 mg/kg i.p., single administration). Urine was then collected over a 24-hour period and blood samples were taken. Biological parameters indicative of renal function were measured and quantification of novel biomarkers of nephrotoxicity was performed using a Multiplex assay. Our results showed that gentamicin at 30 mg/kg and cisplatin at 1 mg/kg had almost no effect on renal function parameters. On the contrary, gentamicin at 100 mg/kg and cisplatin at 5 mg/kg induced a marked alteration of renal function (e.g.
Abstracts
172 Age-related hearing loss in the C57BL/6N mouse: Time course analysis of auditory function with implications for auditory safety study design Matthew Abernathya,b, Rachel Tappa, Joshua Yodera, Andrea Kocha, Carolyn Hedkea, David Gauvina, Theodore Bairda a
MPI Research, Mattawan, MI, USA Western Michigan University, Kalamazoo, MI, USA
An experiment was conducted in guinea pigs in which perilymph samples were collected from three locations along the cochlea, 1) round window, 2) 4 mm from the round window, and 3) apex. The results of this experiment show that larger sample volumes can consistently be extracted from the cochlea when taken 4 mm from the round window. If applied correctly, the information from his experiment can enhance the best practices associated with perilymph collections for auditory safety evaluations.
b
Age-related hearing loss occurs in almost every species, although the time of onset and degree of loss varies from species to species. For instance, guinea pigs generally present with age-related hearing loss at 12 months of age where as some strains of mice experience hearing loss beginning at 6 weeks of age. If a species displaying agerelated hearing loss is used on an auditory safety study, it is important to understand the time course so that the study design can be modified to avoid issues with data interpretation while still assessing safety. An experiment was conducted to establish the time course of age-related hearing loss in the C57BL/6N mouse. This strain was selected as it is commonly used in auditory research and has been shown to develop auditory function deficits early in life. Beginning at 6 weeks of age, auditory brainstem response evaluations were conducted monthly until 21 weeks of age. The cochleae of 6 and 21 week old mice were processed and sectioned for hair cell quantification and histomorphological analysis on other relevant cochlear structures affecting auditory function. The results of this experiment show that the C57BL/6N strain of mouse experiences high frequency hearing loss beginning at 10 weeks of age which progresses to midrange frequencies by 21 weeks of age. Hair cell counts from 21 week old mice showed extensive loss in the basal regions of the cochlea supporting a sensorineural cause for the loss of auditory function. Options for study design modifications are discussed.
doi:10.1016/j.vascn.2013.01.178
173 Influence of collection site on cochlear perilymph sample volume Matthew Abernathya,b, Rachel Tappa, Joshua Yodera, David Gauvina, Theodore Bairda a
MPI Research, Mattawan, MI, USA Western Michigan University, Mattawan, MI, USA
b
In the context of auditory safety evaluations, it is important to assess the ability of a drug to penetrate the tympanic membrane into the middle ear. If a drug does gain access to the middle ear, it is essential to characterize exposure within the cochlea and vestibular system, the inner ear. The relationship between drug exposure and effects on hearing function must be carefully examined to determine a suitable safety margin. The perilymph of the cochlea can be utilized to evaluate inner ear exposure following intra-aural drug administration. It is well known that perilymph drug concentrations, although highly informative, can be capricious due to differences in drug distribution throughout the cochlea. Furthermore, difficulties also arise during the development of analytical methods with the capacity to detect minute concentrations of drug within microliter volume perilymph samples. These potential issues call for the identification of a cochlear site which yields larger sample volumes and, if used as a standard collection site, could overcome variances in drug distribution.
doi:10.1016/j.vascn.2013.01.179
174 Characterization of db/db mice for efficacy/safety pharmacology assessment of antidiabetic drugs Clarisse Duval, Martine Lemaire, Philippe Guillaume, Daniel Provost, Guillaume Froget Porsolt, Le Genest-Saint-Isle, France Pathological models may be relevant and powerful tools for assessing the safety of drug candidates, in parallel of their efficacy, provided they are well-characterized. Our aim was to characterize diabetic (db/db) mice, in comparison with non-diabetic lean controls in respect to their diabetic profile and associated pathological markers. Body weight and basal glycemia were higher in db/db between 7 and 15 weeks of age (34.2 ± 0.3 vs. 18.2 ± 0.2 g and 28.4 ± 1.6 vs. 11.4± 0.6 mmol/l, p b 0.001 at 8 weeks). Food and water intakes over 24 h were increased in db/db (+84% and +162%, p b 0.001). Plasma glucose and insulin were higher in db/db (33.4 ± 1.5 vs. 9.5 ± 0.9 mmol/l and 5.74 ± 0.96 vs. 0.94 ± 0.07 ng/ml, p b 0.001). After oral glucose challenge, glucose increase was relatively higher and sustained in db/db, and returned to baseline within 120 min. Insulin was under baseline from 30 min post-challenge, in contrast with controls being at baseline. Diuresis over 6 hours was higher in db/db (+0.658 g/mouse, pb 0.01). Small intestinal transit was decreased in db/db (−13%, pb 0.05). A tendency towards a decrease was observed on colonic transit. db/db mice showed a moderate hyposensitivity to tactile stimulation (Von Frey: +30%, ns; pinchmeter: +24%, pb 0.01), but a marked hypersensitivity to thermal stimulation (cold plate: −54%, p b 0.001). These findings indicate differences in db/db mice as compared with non-diabetic lean mice, in respect to their diabetic profile but also comorbidity factors. The db/db model is therefore representative of the polymorphism of diabetes, and can be a useful tool for the efficacy/safety pharmacological evaluation of antidiabetic drugs.
doi:10.1016/j.vascn.2013.01.180
175 An evaluation of acute toxicity using the phototactic behavior of Daphnia magna Justin Choi, Rosa Park, Edward Kang, Solomon Oak, Howard Yim, Mark Chung, Hye Sung Kim, Sung Hoon Choo, Jongbin Lee, Andy Suh Prafys Research Laboratory, Englewood Cliffs, NJ, USA Daphnia magna are organisms important for establishing bioassays. They serve crucial roles in pharmaceutical research to help identify chemical compounds that render adverse effects in
Abstracts
ecological environments and to screen new chemical entities for the scientific field. The purpose of this study is to provide scientists with a model that can measure the toxicity of chemicals at the earliest of stages. In this study, a test tube was placed between two light sources, located on opposite ends of an opaque plexiglass box. The illumination of the incandescent light source could be alternatively manipulated according to an experimental schedule, designated to elicit the phototactic responses of Daphnia magna. Under numerous serial dilutions of both ethyl alcohol and sodium chloride – standard chemical compounds employed by scientists – patterns in Daphnia magna phototaxis were used to examine their relationship with the acute toxicity of these compounds. The data were analyzed accordingly by using the unique phototactic index (P.I.), which calculated (a2 + b2 − c2 − d2)/100. The P.I. was plotted with respect to the concentration of the two chemical compounds, in which the slopes were defined to demonstrate the relative patterns in Daphnia phototaxis. Results of a t-test conducted statistically showed that there was a significant difference in Daphnia magna phototaxis in the two compounds, as p = 0.02791. L.J. Whitman & R.J. Miller. (1982). The Phototactic Behavior of Daphnia magna as an Indicator of Chronic Toxicity, Proc. Okla. Acad. Sci. 62, 22–33.
e51
177 Effect of artimisinin compound in experimentally induced colon carcinogenesis Bikash Medhi PGIMER, Chandigarh, India
Investigating an invertebrate model in safety pharmacology using the heartbeat of Daphnia magna Solomon Oak, Rosa Park, Mark Chung, Justin Choi, Edward Kang, Joseph Yang, Daniel Hong, Jamie Han, Nuri Ra, Hye Sung Kim, Sung Hoon Choo, Jongbin Lee, Andy Suh
Artemisinin, a sesquiterpene trioxane lactone had been studied for the chemopreventive efficacy in 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis in rat model. Adult Wistar rats of either sex weighing between 100 and 120 g were assorted into four groups with six animals per group. While group 1 served as control and received the vehicle of the drugs, group 2, 3 and 4 were administered weekly subcutaneous injection of 20 mg/kg DMH for 15 weeks. Group 3 was concomitantly given a weekly dose of 81 mg/kg of 5-FU intraperitoneally and the group 4 concomitantly received artemisinin orally in a dose of 82 mg/kg daily for 15 weeks. Animals were sacrificed at the end of 15 weeks, body weights recorded and the colons were subjected to macroscopic and histomorphological studies. After the experimental period, average number of ACFs/microscopic field and levels of LPO were found to be increased, whereas a significant decrease in the activities of antioxidants (superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione) was observed in DMHtreated rats. Artemisinin administration significantly inhibited the formation of ACFs and decreased significantly the numbers and size of tumors. In addition to this, artemisinin treatment decreased the morphological alterations of ACFs, decreased the levels of LPO and restored the activities of antioxidants to near normal levels. Furthermore artemisinin decreased the levels of pro inflammatory cytokines and induced apoptosis in the colons of DMH treated rats. Thus results obtained in the present study suggest artemisinin as an effective chemopreventive agent on colon carcinogenesis induced by DMH.
Prafys Research Laboratory, Englewood Cliffs, NJ, USA
doi:10.1016/j.vascn.2013.01.183
doi:10.1016/j.vascn.2013.01.181
176
Daphnia magna is an internationally recognized invertebrate model for various toxicological studies and bioassays. Due to the observable heartbeat changes through the transparent carapace, adverse effects of chemical compounds can be reliably detected through bioassay. However, detailed data on the daphnia with incubation duration were scanty and needed to be elaborated. The purpose of this study was to investigate the impacts on heart beat of daphnia by incubation with different duration in either environmental or pharmaceutical compounds. Twenty milliliter for an insecticide and herbicide of concentrations were serially diluted with carbonated filtered water. Six of 2 mL replications of each concentration were made in six test tubes with a micropipette. Daphnia magna were incubated in the test tubes for durations of 0.5, 1, 24, 48 and 72 hours. The heartbeat of Daphnia magna was counted before and after the incubation. For pharmaceutical compounds, dodecyl sodium sulfate and n-Dodecyl b-D-maltoside were tested with the same methods as described. The average change in heartbeat per minute was plotted on a graph with respect to the concentrations. The slope can be used to determine a concentration's effect on viability relative to other concentrations. The change in heartbeat showed a positive correlation with the concentration of the chemical compound. Through this study, establishing the adverse concentrations of these chemical compounds and possible ecological applications could be achieved. doi:10.1016/j.vascn.2013.01.182
178 Deleterious effects of gentamicin and cisplatin on renal function in rats and early detection of drug-induced kidney injury using biomarkers Eric Delpy, Marine Habault, Catherine Le Quement, Christophe Drieu La Rochelle Biotrial Pharmacology, Rennes, France Drug-induced kidney damage constitutes an important cause of acute renal failure and chronic kidney diseases in clinical setting. Unlike other organs, disturbance of kidney function is apparent only when a renal insult is well advanced. Thus, there is a need during safety pharmacology assessment to detect early injury to the kidney. The present work was carried out to evaluate renal function in conscious rats treated with gentamicin (30 and 100 mg/kg s.c. for 7 days) or cisplatin (1 and 5 mg/kg i.p., single administration). Urine was then collected over a 24-hour period and blood samples were taken. Biological parameters indicative of renal function were measured and quantification of novel biomarkers of nephrotoxicity was performed using a Multiplex assay. Our results showed that gentamicin at 30 mg/kg and cisplatin at 1 mg/kg had almost no effect on renal function parameters. On the contrary, gentamicin at 100 mg/kg and cisplatin at 5 mg/kg induced a marked alteration of renal function (e.g.