Developmental and neurobehavioural toxicity study of arsenic on rats ...

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Keywords: Arsenic, Development, Gestational exposure, Neurobehavioural endpoints, Teratology ...... calcium carrageenan, and hydroxyurea, Toxicol Appl.
Indian Journal of Experimental Biology Vol. 50, February 2012, pp. 147-155

Developmental and neurobehavioural toxicity study of arsenic on rats following gestational exposure D N Gandhi1, G M Panchal1 & K G Patel2 Department of 1Neurobehavioral Toxicology and 2Endocrinology, National Institute of Occupational Health (ICMR), Meghaninagar, Ahmedabad 380 016, India Received 24 February 2011; revised 22 November2011 To characterize developmental and behavioral alterations induced by arsenic exposure, Albino rats were exposed to arsenic (0, 1.5, 3.0 and 4.5 mg/kg/day/po) from gestation day 8 to till parturition and the offspring were observed over the first 3 postnatal weeks, until they were weaned on post-natal day (PND) 21. Once the pups were delivered (PND0), the treatment was discontinued. All pups were assessed for physical development, reflex development, strength and motor coordination from standard neurobehavioural developmental test batteries beginning on PND1. Gestational administration of arsenic at tested dose levels, showed no significant changes in the day of appearance of eye opening, startle reflex, negative geotaxis and spontaneous alteration performance in comparison to the control group. The number of live fetuses, mean fetal body weight and percentages of resorptions or malformations per litter were not affected by arsenic exposure. No treatment-related malformations or developmental variations were noted at any exposure level, suggesting that arsenic exposure at this dose level did not adversely affect behavioural endpoints of developmental toxicity. Keywords: Arsenic, Development, Gestational exposure, Neurobehavioural endpoints, Teratology

Arsenic exposure can come from a variety of sources, including diet, drinking water, and both indoor and outdoors residential use. Neurobehavioural evaluations are widely used to examine the potential neurotoxicity of arsenic and other chemicals1-3, since neurobehavioural performance can be a sensitive biomarker of the neurodevelopmental consequences of exposure of environmental agents. There are various methodologies to assess these processes, which depend on factors such as cross species generality and parallels to human behaviour4. Data from animal studies5 demonstrate that arsenic can produce developmental toxicity, including malformation, death, and growth retardation in hamsters, mice, rats, and rabbits. It is common for toxicants that are administered during pregnancy to affect both the mother and the fetus. More detailed information concerning maternal toxicity (food intake, pregnancy weight gain, mortality, gross and histopathology) is available from developmental toxicity studies conducted by gavages6,7. Fetal exposure to environmental —————— *Correspondent author Telephone: 91 079 22686351/52/59 Fax: 91 079 22686110 E-mail: [email protected]

contaminant such as arsenic at apparently non-toxic doses may alter behaviours at adulthood in animals8-11. Some developmental neurotoxicants are structural teratogens as well, but behavioural dysfunctions may be more serious than structural defects under certain circumstances12. Little work has been done on postnatal neurobehavioral endpoints after prenatal exposure13. Though postnatal weight or maturational indices was reported, survival through 30 days of age was reduced in mice treated with sodium arsenite by oral route (5 mg/kg) or ip injection (2 or 4 mg/kg) on gestation day (GD) 1-179. Also, studies8,14 were conducted comparing single dose gavages and ip administration of sodium arsenite. Fetal effects (malformation and embryolethality) were higher by injection than by oralroute8,9,14. In contrast, maternal mortality was higher by the oral route than by injection. Hood et al.14 compared the doses of sodium arsenate considered to produce similar levels of maternal toxicity in mice by gavages (120 mg/kg) or ip injection (40 mg/kg) on single days between GD7 and GD15. With gavages, statistically significant increases were reported for in utero death and resorptions on GD11, and for skeletal malformations on GD9. There was no statistically significant

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increase in gross malformation on any day. For chronic administration, most studies have used oral routes. Arsenic at 0.53, 3.65, or 13.25 mg/kg/day was administered in feed15 in two-generation study and at a dose of 5 ppm arsenite (salt unspecified) in drinking water to mice for three-generations study. Measures relevant to developmental toxicity were average litter size, dead litters, “young deaths”, and “runts”. A large dose response relationships study, for developmental endpoints was described in detail16. In a study to determine the combined developmental toxicity of chromium, copper, arsenates in rats, arsenic (sodium arsenate, 5 mg As/kg, ip, GD8-19) did not influence fetotoxicity (resorption, live fetuses), fetal weight, or maternal toxicity (maternal weight gain) but did significantly increase the incidence of fetal abnormalities as a group (retarded skeletal development, vertebral absence or delayed ossification, short ribs, ectrodactyly, subcutaneous hemorrhage) and of ectrodactyly considered separately17. A characteristic pattern of malformations is produced, and the developmental toxicity effects are dependent on dose, route, and the day of gestation when exposure occurs. Therefore, the present study aims to assess whether in gestational exposure arsenic has a detrimental impact on early physical and neurobehavioural outcomes. Further, an in vivo evaluation of behavioural toxicity on some endpoints has been performed, in order to improve its further developmental effects. Materials and Methods Prenatal arsenic exposure—Time pregnant female prim gravid (GD0) Wistar albino rats (55), aged 7075 days and weighing 175-200 g from the breeding colony of the National Institute of Nutrition, Hydrabad, India were received and acclimatized for a week at NIOH’s animal house in individual cages with bedding and given ad libitum access to food (Purina lab chow) and tap water. National and international guidelines for housing and treatment were followed. The animals were maintained in a temperature-controlled environment (22° ± 2°C) at 70% RH and on a 12:12h light/dark cycle. All experiments were performed between 09.00 and 17.00 hrs. The experimental protocol was approved by the Institutional Animal Ethics Committee and was conducted according to the Indian National Science Academy (INSA) guidelines for the use and care of experimental animals, chemical, dose and treatment schedule.

Out of 55 GD0 females, 39 pregnant females delivered the pups; rest of the 16 females did not deliver. At GD0 females (39) were assigned to one of the following four treatment groups: Gr.1 (As, 1.5 mg/kg; n=8), Gr.2 (As, 3.0 mg/kg ;n =10), Gr.3 (As, 4.5 mg/kg; n=12), or Gr. 4 (Control 0.9% saline water; n=9). Dams were weighed and dosed daily from GD8 to till parturition. Each group was allowed to deliver the pups. Gr.1-4 delivered 62, 89, 89, and 60 pups respectively. For the present study, 20 pups from each treatment groups were randomly selected and evaluated for survival, growth, toxicity, development and behaviours. The day of parturition was defined as postnatal day (PND0), meaning that the maximum resolution for gestational length was one half day. The pups were counted, examined for gross malformations and weighed individually. From PND1-23, pup body weights and maternal behaviour were recorded daily during nursing. The pup was considered the experimental unit. On PND23, pups were sorted on the basis of sex and observed for mortality, signs of toxicity, survival, growth, development and behaviours. On birth (PND0), the numbers of stillborns and live pups in each litter were recorded. On PND1on wards, each litter was examined daily for any changes in appearances, behavioural parameters or survival and all deaths were recorded. Birth measures—Pups were examined on PND1 for morphological anomalies (e.g., missing digits, facial malformations etc.), sexed by relative anogenital distance and culled pseudo-randomly to 20 pups from each group of treatment. Gestation length was calculated at birth and the following litter data were collected on PND1: pup size, sex ratio (as percent males), body weight for each pup and the number of malformed offspring. Pups remained with their biological mothers and postnatal biobehavioural maturation of the pups was assessed over the first 3 postnatal weeks, until they were weaned on PND23. Developmental and behavioural milestones— Twenty pups from each treatment groups were weighed on PND1,4,8,12,16, and 21, and the emergence of physical maturation landmarks including physical developmental parameters such as pinna detachment (PND2-5); incisor irruption (PND6-7); eye opening (PND11-16); development of fur (PND9); ear unfolding (PND2)19,20 testes descendent (PND25)21 and vaginal opening (PND30)21 were carried out at the day of appearance. All pups in each

GANDHI et al.: DEVELOPMENTAL & NEUROBEHAVIOURAL TOXICITY OF ARSENIC

litter were assessed every day even after attaining each milestone. The dam was first removed from the home cage and the individual tests within each testing category proceeded in the following order: (i) reflex development-righting reflex, (ii) motor coordinationthe negative geotaxis test, (iii) muscle strength test, (iv) open field activity and (v) spontaneous or non-behaviour tests. The days of appearance of each milestone were recorded using previous criteria22-25. Reflex development—For the righting reflex, the pup was placed on its back (dorsal position) and the time required (latency(s)) for the animal to turn ventrally itself within 60 sec was recorded23. Each pup received five trials per day from PND1-5. Other reflexes were assessed such as palmer grasps (PND11), negative geotaxis (PND9-11), rotarod (PND20), cliff avoidance (PND1-4), free-fall righting (PND12-14) and auditory startle (PND7) as described26. Also, forelimb placing (PND4-5), hind limb placing (PND6), surface righting (PND9-11), ear twitch (PND18), tail pinch and limb withdrawal reflexes19,27 were assessed. Aerial righting was recorded as the day the animal turned in mid-air to land ventrically after being dropped, back downwards, from 35 cm height on to a cotton-wool pad in three consecutive trials; palmar grasp was evaluated by stroking gently the palm of forepaw with a forceps and observing the digital flexing response. Strength—Rotamax (Columbus Instruments, Columbus, Ohio, USA) was used for assessing forelimb grip strength on PND20. The drum rotated at particular speed until the rat falls of the drum in a 30 sec session. Latency to escape from the drum and percentage of animals that fell from the drum were calculated and compared between groups28. Pups’ forelimb grip strength was also assessed using a hanging grip test, measuring the length of time the pup could hang from a 2.2 mm diameter bar, and suspended 12 cm from a surface covered with bedding. The hanging time was assessed in one trial (30 sec) each on PND12 and PND16 immediately following the second trial in the forelimb grip test. Motor and coordination—Motor and coordination capacity was examined using the negative geotaxis test as described on PND9-1123. On day 20 the animal’s ability to hang, the length of time it does hang, and its activity while hanging were observed29. A 17 mm diameter wooden rod is placed in the center of a container filled with water at 15°C. The rat is placed at the base of the rod and must climb to the

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platform within 180s28. A 15 mm diameter rope is held taut in a vertical position. The rat is placed at the top of the rope and must descend the rope to a sawdust-filled box at the bottom. Distance traveled (cm), time limitation(s), cling time(s), and frequency of falling were monitored30 and recorded. Activity and emotional reactivity: exploratory behaviour in an open field (the top of a laboratory cart, size 42 × 42cm)—The rat explored an open field24,25 (the top of a laboratory cart) for 3 min, the observed activity level, and recorded the number of head elevation, hind limb elevation (PND-11), and numbers of lifting of the hindlimbs and pelvis, rearing (no of rare), pivoting (circular movement, PND2-12), grooming, sniffing, biting and licking. The auditory startle (PND7) was recorded as the day animal responded with a sudden and brief extension of the hindlimbs to the sound of a clicker or snapping19,23,31. This behaviour is generally recorded during 30sec observation time. Gait abnormality (walking pattern) was observed. Spontaneous or non-behaviour tests—Animals were tested for spontaneous alteration on PND17. The apparatus was eight-arm radial maze converted in to a T-maze (a 16.5" base and two arms each 16.5" (total 33.0") long along with central arena 10.8" wide with transparent lid. Two emitter detector infrared beam pairs per arm to monitor animal behavior (Columbus Instruments, Columbus, Ohio, USA). Latency to enter one arm of the maze, and arm entered recorded. The animals were tested in consecutive trails (intertribal interval 20s) until they entered the contra lateral arm, with a maximum of 5 trials. When the latency to enter one of the sides was longer than 300s, the animal was excluded then subsequent testing. Percentage of failure to alternate was also calculated for each pup and compared between groups. Maternal behaviours—Maternal behaviours were observed daily in the home cage of each dam and her pup between gestational day 21 and PND1 to 14. Maternal behaviour parameter indicates mother-pup relationship. Usually control mother spent more time involved in the pup-directed behaviours of nursing and licking and less time in nest building in first two postnatal weeks. The maternal behaviours were assessed by observing lactating females in their home cage during an observation period of 80 min on PND2-15. Each dam was observed once every 4 min for total 20 observations. During each 4 min

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observation period, the experimenter recorded which behaviour the lactating female was displaying at the moment of observation. The maternal behaviours monitored were as follows: (a) in nest (home cage), (b) number of pups nursing, (c) number of pups not nourish, (d) number of pups with mother, (e) number of pups alone, (f) number of pups with other pup, (g) licking pups, (h) eating/drinking, (i) grooming, (j) resting and (k) forced nursing. Statistical analysis—All data are expressed as mean ± SE. Data were statistically analyzed using multivariate analysis of variance (ANOVA). The litter was considered as the analysis unit. The level of statistical significance was set at P