INTRODUCTION:
Uranium is a heavy metal which can be used as an abundant source of concentrated energy. Uranium occurs in most rocks in concentrations of 2 to 4 parts per million and is as common in the Earth's crust as tin, tungsten and molybdenum. Uranium is predominantly present in aqueous system as U (IV) and U (VI). U (IV) is highly insoluble and immobile and U (VI) is highly soluble and mobile[1]. The toxicity of uranium varies according to the chemical form exposed, route of exposure, age, sex, body mass index and species[2]. More soluble uranium compounds have highest toxicity.
The spectrum of acute toxicity has been extensively studied in animals and to some extent in humans. Human beings are constantly exposed to a certain amount of uranium because of its heterogeneous presence in natural form in the environment. Inhalation, ingestion or skin contact leads to respiratory diseases, kidney failure, reproductive diseases .
Acute exposure or long term to uranium affects kidney, liver, gastrointestinal tract, bones . and the most ingested uranium is excreted in urine[3,4]. Regarding the information on toxicity, health hazards of a compound LD50 data is very important. Various accepted methods[5-10] are used to calculate LD50. However, literature provides only less information regarding the LD50 of uranium compounds. The Oral and subcutaneous LD50 of uranyl acetate in Sprague Dawley rat is reported as 242 and 8.3 mg/kg bdwt[11] but satisfactory data related to LD50 of uranyl nitrate in mice is not available. Differences in LD50 in different species might give informations or clues to difference in absorption, metabolism and mechanism of toxicity of a substance[12]. Hence the present study aims to determine the LD50 of uranyl nitrate via intraperitoneal route in male Swiss albino mice.
MATERIALS AND METHODS:
Male Swiss albino mice of 8–10 weeks of age, weighing approximately 25 ± 5g were selected for the study from an inbred colony maintained under the controlled conditions of 23 ± 20C, relative humidity of 55 ± 5% and 12 h photoperiod with sterile food and water. Experiments were conducted after obtaining approval (No.:1/2014) by the Institutional animal ethics committee. The animal care and handling were according to the Institutional guidelines for animal experimentation and as per the recommendations of OECD guidelines[13] .
Preparation of uranyl nitrate dose and administration was done as follows, Animals were fasted prior to dosing, following the period of fasting, the animals (Swiss albino mice) were weighed and treated with a single intraperitoneal dose of uranyl nitrate hexahydrate (MW 502.13) dissolved in 0.9% saline. The volume of dose in rodents should not exceed 1ml/100g b.w[14]. An approximate LD50 can be initially determined as a pilot study by a so called ‘staircase method’ or “up and down” method using a small number of animals (2 each dose) and increasing the doses the test compound[15,16]. Published lethality study on uranium compounds were used to estimate the initial dose for the pilot study. Determination begins with a dose of uranyl nitrate used. The survival or death of the animal at that concentration or dose decides for the next dose either to be increased or decreased accordingly. Five doses were chosen 64, 32, 16, 8, 4 mg/kg bdwt of uranyl nitrate and given intraperitonially to five separate groups and one control group (0.9% saline) five mice in each group,. The animals were observed for a period of 14 days. The percentage of mortality was calculated using graphical method of Miller and Tainter (1944). Body weight of the animal was recorded after the administration of uranyl nitrate.
RESULT AND DISCUSSION:
Acute
lethal dose (LD50) was determined by injecting increasing doses of
uranyl nitrate intraperitoneally in different groups of swiss albino mice and
observed for survival up to 14 days.
Death was observed for all the animals in the group that received 64 mg/kg of
uranyl nitrate. All the animals in the group that received 4mg/kg bdwt survied.
The animals exhibited chewing, licking, labored breathing, gasping and finally
death within 5 days. The number of deceased male swiss albino mice was recorded
for each dose level of Uranyl nitrate shown in (Table 1).The percentage
mortality at each dose level were converted to probit using Finneys method[8]
(Table 2). The percentage dead for 0 and 100 were corrected prior the
determination of probits. Calculation for 0% dead : 100(0.25/n) and for 100%
dead: 100(n-0.25/n) respectively, (n=5). LD50 value of Uranyl
nitrate was determined by the graph plotted between probit Vs log dose, the
dose at probit 5.0 that is concedered as 50% mortality (Fig. 1). Approx. Standard error (S.E) of LD50 is
determined using the equation given below, where N is the number of animals in
each group [17],
. The probits of 84 and 16 were 5.99 and 4.01 (approximately 6
and 4), respectively. The log LD values corresponding to probit 6 and 4 were
obtained from the line of probit versus Log dose graph. In the present case
values were found to be 1.6 and 0.85 and
antilog of these values were 39.8 and 7.07 respectively, the values were
substittuted to the to equation to obtain S.E . In the present study, for male
swiss albino mice Log LD50 is 1.24 and calculated LD50
is 17.37 mg/kg. Standard error obtained from the equation found to be
6.54. Thus LD50 of intraperitoneal route is 17.37 ± 6.54. LD50
concentration of Swiss albino mice treated with uranyl nitrate were calculated
by plotting of probit obtained aganist log dose of uranyl nitrate by a simple
regression plot using Excel (Microsoft.Inc).
Table 1: Graphical method for uranyl nitrate LD50 determination
|
Dose of uranyl nitrate (mg/kg) |
Log dose |
No: of animals died |
Percentage of dead (%) |
Corrected Percentage (%) |
Probits |
|
64 |
1.806 |
5 |
100 |
95 |
6.64 |
|
32 |
1.505 |
4 |
80 |
80 |
5.84 |
|
16 |
1.2 |
2 |
40 |
40 |
4.75 |
|
8 |
0.9 |
1 |
20 |
20 |
4.16 |
|
4 |
0.602 |
0 |
0 |
5 |
3.36 |
The acute LD50 toxicity test is performed in rodents as apart of safety assessment of many substance. Variation in LD50 exists due to difference in factors such as fasted or non fasted, type of diet, route and rate of administration age, sex, purity of substance, number of animals used and length of time the animals[18]. Acute toxicity on soluble uranium compounds reported the oral and subcutaneous LD50 of uranyl acetate in mice as 242 mg/kg and 20.4 mg/kg bdwt[11]. It has been reportted that 5g be provisionally considered the acute oral LD50 for uranium in humans and 1.0 g of uranium is proposed as the provisional acute inhalation LD50[19] .