Insecticides, a class of pesticides, have been extensively used in farming to protect crops against insect pests¹. Despite offering significant benefits of improved crop production, the over-use of pesticides has raised public health concerns globally. Irrational use of pesticides has resulted in environmental contamination and impaired ecosystem harmony by affecting non-target species². Furthermore, pesticide residues are reaching the food chain of man and animals through agriculture products. The consumption of such pesticide residue-laden food is implicated in causing chronic and degenerative diseases in the long run³.

Triazophos is a widely used organophosphate insecticide in agriculture as a foliar spray against a wide variety of insect pests⁴. It is classified by the World Health Organisation as a highly hazardous pesticide (Class Ib) with an oral LD₅₀ of 84 mg/kg body weight in rats⁵. The residues of triazophos present a threat to human life and have recently been reported in various foodstuffs⁶. Multiple studies have reported the toxic potential of triazophos on the liver, kidneys, and brain of rats by altering the oxidant-antioxidant balance^7-9. However, the data on evaluating the effect of triazophos on hemato-biochemical parameters in rats is lacking and forms one of the objectives of our present study.

Quercetin is a naturally occurring dietary bioflavonoid present in fruits and vegetables¹⁰. It is known to exhibit anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-aging, and anti-microbial properties, as demonstrated by various studies. Its free radical scavenging and metal chelating qualities offer benefits in various degenerative disorders¹¹. Besides, it has also been shown to protect experimental animals from pesticide induced toxicity, including organophosphate insecticides^12-13. Regardless of these advantages, the use of quercetin in clinical practice suffers a significant drawback because of its poor oral bioavailability. Various researchers have suggested nanosizing of quercetin to overcome pharmacokinetic issues¹⁴. Hence, our second objective focused on the comparative evaluation of the possible protective effect of quercetin and nano-quercetin against triazophos-induced hemato-biochemical alterations in Wistar rats at the end of 60 days exposure period.

Hematological alteration serves as an essential marker in assessing the toxicity and pathological consequences of any chemical to animals or humans. The chemical may directly alter the cellular constituents of blood or indirectly interact with them through the production of reactive intermediates¹⁶. In the present investigation, triazophos treatment for 60 days noticeably raised the leucocyte count in a dose-dependent way. Quercetin and nano-quercetin reversed this triazophos-induced elevated leucocyte count. Similar to our findings, triazophos at sub-lethal concentration increased WBC count in Channa punctatus¹⁷. Nevertheless, in contrast to our results, triazophos-induced leucopenia was reported previously in Japanese quail¹⁸. The anti-inflammatory action of quercetin might be responsible for reversing the elevated leukocyte counts, as demonstrated by earlier workers¹⁹. Triazophos in our study increased the lymphocyte count in low and high-dose group rats that was brought back to nearly normal values by co-administration of quercetin and nano-quercetin with high dose triazophos. In agreement with our findings, the lymphocyte count was the most affected by triazophos in C. punctatus¹⁷. Ghaffar and co-workers also recorded elevated lymphocyte counts in triazophos treated birds at different intervals and doses used in that particular study¹⁸. However, in a study with buffalo calves, significant lymphocytopenia was reported by triazophos oral dosing for 21 days²⁰. In a recent investigation, low doses of malathion, an organophosphate similar to triazophos, induced proliferation of human lymphocytes without causing cytotoxicity. Malathion upregulated specific genes responsible for lymphocyte multiplication²¹. A similar type of gene regulation might be responsible for the elevated lymphocyte count in our study with triazophos.

Further, quercetin is an antioxidant flavonoid with an established free radical scavenging property. It decreases the overall oxidative stress and protects the cell against DNA damage due to reactive oxygen species. In human lymphocytes, quercetin successfully prevented the chemically induced DNA damage and protected lymphocytes from a proliferative response to exhibit anti-carcinogenic properties²².A comparable protective effect of quercetin might be the reason for reversing elevated lymphocyte count with triazophos in our study.

We encountered a decrease in platelet count in a dose-dependent manner with the administration of triazophos at 1/10^th LD₅₀ and 1/20^th LD₅₀ in rats. Besides, quercetin and nano-quercetin dosing protected the rats against the triazophos-induced drop in platelet count. The literature relating the effect of triazophos exposure and platelet count is limited. Quercetin exhibited platelet-enhancing properties in rats exposed to polychlorinated biphenyls (PCBs)²³. In another study, quercetin ameliorated the decreased platelet count seen with carbendazim fungicide administration in rats²⁴. Other hematological parameters like total erythrocyte count, hemoglobin levels, and hematocrit values did not differ significantly between triazophos treated and control rats. Contrastingly, triazophos administration in Japanese quails led to hypochromic anemia characterized by decreased RBC count, hemoglobin levels, and MCHC.¹⁸ It also significantly decreased the erythrocyte counts, hemoglobin levels, MCHC, MCV, and PCV in C. punctatus¹⁷. The work with triazophos in buffaloes revealed no significant change in ESR or PCV at both doses employed, and hemoglobin levels did not change at a low dose compared to control buffaloes²⁰. This varying response in hematology with triazophos in different species is a matter of consideration for further research.

Our findings revealed no significant differences in the levels of total protein, albumin, globulin, or A: G ratio between control group rats and other groups. In freshwater fish, Labeo rohita, triazophos treatment at sub-lethal concentrations significantly decreased serum protein levels²⁵. The dissimilarity in results could be because of the differences in the dose of triazophos and species tested. Triazophos treatment in our work significantly elevated the blood glucose levels of rats compared to control rats. Nano-quercetin and quercetin dosing considerably brought back the elevated glucose levels to normal in triazophos treated rats. Our data regarding triazophos treatment agrees with a study in White leghorn cockerels, where glucose levels were elevated with triazophos administration. Further, the surge in glucose levels was normalized using cow urine distillate in the study with White leghorn cockerels²⁶. Triazophos also increased the glucose content of Nilaparvata lugens insects by altering the glycometabolism in these species²⁷. Nevertheless, contrary to our findings, triazophos administered orally at 1/500^th LD₅₀ and 1/50^th LD₅₀ dose for 24 weeks in rats significantly increased insulin sensitivity and decreased glucose levels²⁸. Also, triazophos treatment in Perna viridis (sea mussel) altered energy metabolism in ovaries and testes by decreasing the glucose levels in gonads²⁹. The anti-diabetic and glucose-reducing ability of quercetin is well established through various studies³⁰. Quercetin alleviated imidacloprid-induced hyperglycemia in rats and protected them against the biochemical toxicity of the pesticide³¹. Quercetin acts through various targets in the small intestine, liver, pancreas, skeletal muscles, and adipose tissue and regulates glucose metabolism in the body, as demonstrated by the previous studies³².

In our study, nano-quercetin showed better efficacy than quercetin in reversing the hemato-biochemical alterations produced by triazophos. This can be attributed to the increased bio-availability of nano-quercetin as compared to quercetin in its native form. In agreement, previous studies with nanoformulations of quercetin have also demonstrated a more significant protective effect than quercetin against chemical toxicities in various systems^33-34.

The oral LD₅₀ of triazophos as determined in our laboratory using male Wistar rats was found to be 76 mg/kg body weight. Treatment of rats for 60 days at 1/10^th LD₅₀ and 1/20^th LD₅₀ of triazophos produced hemato-biochemical toxicity. Triazophos treatment significantly elevated the total leukocyte count and lymphocyte counts, accompanied by a marked decrease in platelet count and blood glucose levels. Co-administration of quercetin and nano-quercetin with high-dose triazophos reversed the altered hemato-biochemical parameters. Nano-quercetin was more effective than quercetin in protecting against the toxic effects of triazophos, possibly due to an improved pharmacokinetic profile.

Values (Mean±SEM, n=6) bearing superscripts are significantly different in Tukey’s multiple comparison test.

Comparison with the control group and 1/10^th LD₅₀ group are shown by * and #, respectively.

The number of superscripts indicates the level of significance. P<0.05, P<0.01, and P<0.001 are represented by one, two, and three superscripts, respectively.

Values (Mean±SEM, n=6) bearing superscripts are significantly different in Tukey’s multiple comparison test.

Comparison with the control group and 1/10^th LD₅₀ group are shown by * and #, respectively.

The number of superscripts indicates the level of significance. P<0.05, P<0.01, and P<0.001 are represented by one, two, and three superscripts, respectively.

References