1. Introduction
Many studies [
1,
2,
3,
4] have reported toxic and carcinogenic effects induced when humans and animals are exposed to certain heavy metals. Detailed studies have shown that metals like iron, copper, cadmium, chromium, mercury, nickel, and vanadium possess the ability to produce reactive oxygen species (ROS), resulting in lipid peroxidation, depletion of proteins, and many other effects [
5]. Potassium dichromate, which is a form of hexavalent chromium, has been demonstrated to induce toxicity associated with oxidative stress in humans and animals [
6,
7]. Chromium (Cr) is a naturally occurring element found in rocks, animals, plants, soil, and in volcanic dust and gases [
8,
9]. It comes in several different forms including trivalent chromium and hexavalent chromium or Cr(VI). It is widely used in various industries, including pigments for manufacturing and painting, metal plating and leather tanning. Cr(VI) ingested with food, such as vegetables or meat and water, is reduced to Cr(III) before entering the bloodstream [
10,
11]. Chromium enters the body through the lungs, gastrointestinal tract, and to a lesser extent, through the skin [
12,
13].
It is known that oral intake, including food and water, is the major route of exposure to chromium for the general population. Regardless of the route of exposure, Cr(III) is poorly absorbed, whereas Cr(VI) is more readily absorbed [
14,
15]. During reduction process, Cr produces ROS [
16], and generates oxidative stress (OS). Multiple studies of the developmental toxicity of Cr(VI) in experimental animal models, such as rats and mice, have demonstrated a decrease in the viability of the conceptus, both pre- and post-implantation resorptions, decrease in fetal weights and crown-rump lengths, changes in placental weights (decrease or increase), and increase in frequencies of external and skeletal anomalies [
17,
18,
19,
20,
21].
The toxicity of potassium dichromate has not been well-studied in farm animals over the years, and therefore the scarcity of studies confirming its effects on these animals have revealed the need to carry out toxicity studies on it. Thus, the objective of the present work was to evaluate the oxidative effects of potassium dichromate on biochemical, hematological, and reproductive parameters in female rabbits.
2. Materials and Methods
2.1. Animals
Twenty-eight adult does (New Zealand breed) of 6 months old, weighing 2.8–3.0 kg, reproduced at Teaching and Research Farm of the University of Dschang, were used. The animals were maintained individually in cages of wire netting (96 cm long, 40 cm wide, and 15 cm high) and in galvanized metal, forming a battery of cages. These cages were each equipped with an eater and a drinker. The animals received water and feed
ad libitum. The composition and chemical characteristics of this feed are summarized in
Table 1. Experimental protocols used in this study were approved by the Department of Animal Science, FASA, University of Dschang - Cameroon and strictly conformed with the internationally accepted standard of ethical guidelines for laboratory animal use and care as described in the European Community guidelines; EEC Directive 86/609/EEC, of the 24 November 1986.
2.2. Chemicals
Potassium dichromate (K
2Cr
2O
7) is a compound that is prepared from chromite (FeCr
2O
4) and is also called chrome in iron ore. Its atomic weight is 51.996 [
22]. This chemical was obtained from Sigma Aldrich, Berlin, Germany.
2.3. Toxicological Assessment
Twenty-eight animals were divided into four groups of seven females, with comparable average body weight. Group A (the control) received 1 mL of distilled water, while groups B, C, and D received doses of 10, 20, and 40 mg/kg body weight (bw) K2Cr2O7, respectively, via gavage for 28 days. Rabbits were observed during the first 24 h for the beginning of any immediate toxic signs and daily for 28 days. At the end of the experiment, animals were anesthetized with ether vapor.
2.4. Hematological and Biochemical Analysis
Blood samples were obtained by cardiac puncture and collected without anticoagulant for biochemical dosages and with anticoagulant (EDTA) for complete blood count. The biochemical parameters analyzed from serum were total cholesterol (TC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea (Ur), creatinine (Cr), Albumin (Al), and total protein (TP), performed with appropriate commercial Chronolab kits (Barcelona, Spain). The spectro-photometric method was used according to kits instructions. The hematological parameters analyzed were white blood cells (WBC), lymphocytes (LY), monocytes (MO), granulocytes (GR), red blood cells (RBC), platelets (PLT), plaquetocrit (PCT), hematocrit (HCT), and hemoglobin (Hb), performed using a veterinary hematology analyzer Genius KT 6180 (Shenzhen Genius Electronics Co., Ltd., Hong Kong, China). Follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were dosed in serum and in homogenates of ovary. This dosing was realized with the help of AccuDiagTM ELISA kits from OMEGA Diagnostics Ltd (Alva, United Kingdom) respecting the immuno-enzymatic method.
2.5. Weight and Volume of Organs
After animal sacrifice the right ovary, kidney, liver, and uterus were removed and weighed.
2.6. Oxidative Stress Markers
Activities of superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), and concentration of malondialdehyde (MDA) in liver, kidney, ovary, and uterus were measured.
2.6.1. Estimation of Catalase Activity
Catalase activity was estimated according to Aebi [
23], depending on the ability of H
2O
2 to decompose via the action of CAT to produce H
2O and O
2. The decrease in the absorbance in the UV region per time is corresponded to CAT activity. A volume of 2.0 mL of substrate (10 pmol/mL of H
2O
2 in 50 mmol/L sodium-potassium phosphate buffer, pH 7.0) was incubated with 100-µL serum. The decomposition of H
2O
2 was followed directly for 2 mins by the decrease in absorbance at 240 nm.
2.6.2. Estimation of Glutathione
Glutathione level was measured according to the method of Moron et al., [
24] based on the reaction of GSH with 5,5-dithiobis-2-nitro-benzoic acid (DTNB)(Sigma Aldrich, Berlin, Germany) at pH 8.0 was added to the tubes and the intense yellow color formed was red at 412 nm in a spectrophotometer after 10 min. A standard curve of GSH was prepared using concentrations ranging from 2 to 10 nmol of GSH in 5% trichloroacetic acid (Labtech, Windsor, Australia).
After centrifugation, the absorbance of yellow color was measured and the results were calculated from the glutathione standard curve.
2.6.3. Estimation of Lipid Peroxidation
Lipid peroxidation was estimated by reaction of thiobarbituric acid (TBA) (from Qulaikems, New Delhi, India) with malondialdehyde (MDA) according to Botsoglou et al., [
25]. In the presence of an acid and heat (pH 2–3, 100 °C), MDA condensed with two molecules of TBA to produce a pink color complex that absorbs at 532 nm. A total of 105 μL of orthophosphoric acid at 1% and 500 μL of the precipitation mixture (1% TBA in a 1% acetic acid solution) was added to 100 μL homogenate. The mixture of each tube was homogenized and placed in a boiling water for 15 min. The tubes were cooled in an ice-bath and the mixture was centrifuged at 3500 rpm for 10 min. Absorbance was read at 532 nm against the control.
2.6.4. Estimation of Superoxide Dismutase
Adrenaline is stable enough when pH is acidic. When pH increases, the rate of auto-oxidation of adrenaline increases. The dosage of SOD is thus based on the capacity of SOD to inhibit or slow down auto-oxidation of adrenaline to adreno-chromium in a milieu of a base. The method proposed by Misra and Fridovich [
26] was used in this study. Microtubes of serum were introduced into the spectrometer, as well as 1660 µL of carbonate buffer solution (pH = 10) and 200 µL of adrenaline (0.3 mM). The absorbance of adreno-chromium formed was read at 480 nm 30 and 90 s after the initiation of the reaction.
2.7. Statistical Analysis
Data were submitted to analysis of variance (ANOVA) one factor to test the effect of K2Cr2O7 on the studied parameters. Duncan’s test was used to separate means when there was a significant difference. The results were expressed in the form of mean ± standard deviation. The limit of significance was set at 5% and the software IBM SPSS Statistics 20.0, (Armonk, NY, USA) was used for analysis.
4. Discussion
The exposure of animals to heavy metals can lead to adverse effects on their health in general and on their reproduction in particular. Kidney and liver are organs very important in the evaluation of the toxic potential of a substance [
27]. They are associated with the metabolism and excretion of toxic substances [
28] such as heavy metals. In the current study, live body weight decreased significantly with increasing doses of potassium dichromate throughout the experimental period. This result is in accordance with those of [
29,
30]. This may be due to the effects of potassium dichromate on female rabbits. The weight and volume of kidney and liver, as well as ovary weight, were comparable among treatments. This observation could signify that the dose of potassium dichromate administered did not have a pronounced effect on the anatomy of those organs. These results do not agree with Saha et al. [
31], who recorded a significant reduction in the weight of the reproductive organs along with an increase in the weight of the liver and kidney; and Petrovici et al. [
32], who noted an increase in ovary weight of rats exposed to potassium dichromate at a dose of 10 mg/kg bw with respect to the control.
Kidneys perform two major important functions: first, they excrete most of the end products of bodily metabolism, and second, they control the concentrations of most of the constituents of the body fluids [
33]. As markers of renal function, uric acid, urea, and creatinine are routinely used for analysis. In kidneys, urea is filtered out of blood by glomeruli and is partially reabsorbed with water [
34]. Creatinine is a breakdown product of creatine phosphate in muscle and is usually produced at a constant rate by the body depending on muscle mass [
35]. The biochemical analyses in this study showed an increase in the concentrations of creatinine, urea, and total cholesterol levels, with a decline in proteins and albumin levels. Albumin is a specific protein and its decrease in this study can be associated with the decrease in proteins. This decrease reflects damage in hepatocytes and indicated the general and systemic toxic effect of heavy metals on rabbit does [
36]. Similar results were reported by Saha et al., [
31] in rats treated with potassium dichromate. An increase in cholesterol level in this study might be due to less utilization of these nutrients at the tissue level [
37]. The increase in creatinine and urea could be due to the dysfunction of glomerulus, which are the structures responsible for the renal filtration. That of urea could also be explained by the increase in proteins’ catabolism, due to the high synthesis of the enzyme arginase, which intervenes in the urea production [
38]. Transaminases and lactate dehydrogenase are the most sensitive biomarkers directly implicated in the extent of cellular damage and toxicity because they are localized in the cytoplasm and are released into the circulation after cellular damage [
39].
A high level of serum of enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicate liver damage, such as that due to viral hepatitis, as well as cardiac infraction and muscle injury. Serum ALT catalyzes the conversion of alanine to pyruvate and glutamate. Therefore, serum ALT is more specific to the liver, and is thus a better parameter for detecting liver injury [
40]. Their increase in the current study suggests that potassium dichromate could have generated reactive oxygen species, thus oxidative stress leading to hepatotoxicity and nephrotoxicity. This may be due to the impairment in their synthesis or poor liver function associated to oxidative stress [
41]. Similar results were observed by Abbas and Ali, Zhu et al., Mehany et al., Mohamed and Saber, Saha et al., and Krim et al. [
31,
42,
43,
44,
45,
46] in rats treated with potassium dichromate.
Blood acts as a pathological reflector of the status of exposed animals to toxicants and other conditions and/or agents [
47]. Monocytes, granulocytes, and lymphocytes, which are associates of white blood cells, act as defenders against pathogens and tend to increase in case of infection. Thus, the increase in white blood cells in this study can be explained by the fact that potassium dichromate was recognized as a toxicant. Hemoglobin is related to the total population of red blood cells in blood. The decrease in hemoglobin suggests that potassium dichromate could have blocked the erythropoietin release, which is the humoral regulator of red blood cell production. The similar decrease in platelets can be attributed to the blockage of thrombopoietin. The present results are in agreement with the findings of Shrivastava et al., Stana et al., Ounassa, Vihol et al. [
48,
49,
50,
51] in rats and mice treated with potassium dichromate.
The hypothalamic–pituitary–gonadal (HPG) axis plays a critical role in the control of reproduction. Potassium dichromate exposure led to the decrease in follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol. This might be due to the decrease in protein levels. In fact, FSH and LH are hormones synthesized from proteins captured from blood. Thus, the reduction in total proteins in blood could have led to a decrease in FSH and LH concentrations in potassium dichromate-treated rabbit does. The abnormal levels of sex hormones recorded in this study are in accordance with those registered in female rats exposed to potassium dichromate, by Assasa and Farahat [
52] suggesting a disruption of steroidogenic function. Animals in this study are of the same age and in principle age is not therefore a factor of variation in hormonal level in this case. Considering other studies, such as that carried out by Zhang et al. [
53] in New Zeeland female rabbits and Labib et al. [
54], age and corpus luteum formation can be a factor of hormonal expression.
The changes in oxidative stress biomarkers have been reported to be an indicator of a tissue’s ability to cope with oxidative stress [
55]. The antioxidant enzyme catalase (CAT) acts as defence against free radicals. It is responsible for the catalytic decomposition of hydrogen peroxide to molecular oxygen and water. Glutathione (GSH) is normally present in millimolar concentrations in cells and is known to protect the cellular system against the toxic effects of lipid peroxidation. It is very important in maintaining cellular redox status [
56] and its depletion is considered as a marker of oxidative stress [
57]. The decreased superoxide dismutase (SOD) activity may lead to massive production of the superoxide anion. The production of such anions overrides enzymatic activity and leads to a fall in its concentration in renal tissue. It was indicated by Srinivasan et al. and Pedraza-Chaverri et al. [
58,
59] that most of the antioxidant enzymes become inactive after potassium dichromate exposure, either due to the direct binding of heavy metals to enzyme active site or to the displacement of metal co-factors from active sites. Increased lipid peroxidation is indicated by the increase in malondialdehyde (MDA), which is an end-product of lipid peroxidation. The current results showed that treatment with potassium dichromate induced oxidative stress notified by a significant decrease in GSH content, SOD, and CAT activities, and a significant increase in MDA level, as compared to control values. The results obtained for the various oxidative stress biomarkers in the present study reflect those obtained by Mehany et al. [
44], Shati [
60] and Mohamed and Saber, [
45].
5. Conclusions
In conclusion, the present study proved that potassium dichromate administration to does led to a significant increase in the levels of creatinine, urea, alanine aminotransferase, and total cholesterol, with a significant decrease in renal tissue proteins, hepatic tissue proteins, and albumin levels, as compared with the control group. This may be evidence for its hepatotoxic and nephrotoxic effects.
A significant decrease in follicle stimulating hormone, luteinizing hormone, and estradiol levels was equally observed in potassium dichromate-treated does with reference to the control females, indicating its dangerous effects on reproductive hormones.
The administration of potassium dichromate brought about a significant increase in white blood cells and lymphocyctes, with a significant decrease in hemoglobin levels with respect to does that received distilled water only. This could be a confirmation of its hematotoxic activities in female rabbits.
Does treated with potassium dichromate only registered a significant decrease in glutathione, superoxide dismutase, and catalase activities, while malondialdehyde increased significantly relative to the control does. This could be attributed to its deleterious effects on enzymatic antioxidants and its lipid peroxidation activities. The observed effects of potassium dichromate in this study showed that it had dose-dependent intoxication.