Biochemical and Histological Evaluation of Penoxsulam Herbicide on an Animal Model

Aims: Many herbicides react to the body system and might effect the activity of hormones in the human body. The present work aims to determine the potential impact of 90 days of repeated exposure to Penoxsulam herbicide by oral gavage on the Liver, Kidney, Thyroid endocrine profile and biochemical stress in the wistar rat model. Study Design: The primary study was performed to Wistar rats grouped into 6 groups. Four groups were picked for low dose, middle dose, high dose, and high recovery dose, respectively. They were administered the Penoxsulam at dose levels of 100, 300, 500 mg/kg body weight. Similarly, 2 recovery groups were classified as control and recovery control groups, and doses were administered to them only through the corn oil (vehicle) via the oral route with the help of a suitable cannula for 90 days. Place and Duration of Study: Toxicology department, Shriram Institute for Industrial Research, Delhi (INDIA), July 2020 and June 2021. Methodology: In this study, healthy 60 male and 60 female Wistar rats aged 6-8 weeks, weighing 130-190 gm, were used. Before commencing the study, permission from IAEC (Institutional animal ethics committee) was taken for this experiment (CPCSEA). Results: This study evaluates significant changes in the body weight of rats; moreover, Original Research Article Chaurasia et al.; JPRI, 34(4B): 12-23, 2022; Article no.JPRI.79828 13 Penoxsulam elevated the significance level of SGOT, SGPT, BUN, Urea, and Creatinine. No alterations were seen in Hematology parameters and Ophthalmology examination. Also, physiological changes were examined after exposure to penoxsulam in rats. Conclusion: Therefore, Penoxsulam showed harmful toxic effects on the Kidney and Liver. However, no alteration has been seen in the thyroid profile (T3 triiodothyronine, T4 thyroxine, TSH thyroid-stimulating hormone) of Wistar rats during the experimentation period.


Penoxsulam
(2-(2,2-difluoroethoxy)-N-(5, 8-dimethoxy1,2,4] triazolo [1,5-c] pyrimidin-2-yl)-6-(trifluoromethy) benze-nesulfonamide)) [1] is a new triazolopyrimidine sulfonamide herbicide used in California for post-emergence, broadspectrum weed control in rice (Oryza sativa L.). Penoxsulam is a systemic herbicide that internally disrupts growing weeds, being absorbed by the phloem and xylem tissues, leading to death. Penoxsulam is used to control broadleaf, sedge, and grass weeds in transplanted, dry-seeded, and water-seeded rice [2]. Adverse effects on plants by Penoxsulam depend on the defoliant metabolic process rate in plants and not only on their inhibitory strength upon the target site [3]. This systemic herbicide is absorbed primarily via leaves and secondarily via roots translocated in both xylem and phloem. Weeds treated with Penoxsulam will stop growing almost immediately. The signs show rapid growth obstruction and a chlorotic growing site with the death of the terminal bud, resulting in the end of sensitive weeds in two to four weeks. Its primary use would be in a postemergence implementation in parched seeded, water-seeded, and transplanted rice [4][5][6][7][8][9][10][11][12][13][14][15][16]. Rice is an essential food in one's daily life, and 90% of the world's population depends on it. Nowadays, herbicide sprays are widely used for weed control, and they are very harmful both to humans and animals.
Farmers working with Penoxsulam can also be exposed to the herbicide in the course of maintenance, sampling, trials, or any other process [17] Normally farmers are exposed to herbicides via inhalation, ingestion or topically, and these herbicides are mainly absorbed in the blood stream to damage the organs. The hepatorenal are major target organs which plays an important role in purifying the organisms, via absorption and elimination of the chemicals and its consumption. These two organs are more sensitive for enhance toxic effects by chemicals as herbicides [18,19,20,2,[4][5][6][7][8][9][10][11][12][13][14][15][16][21][22][23][24][25]. Hepato-renal changes after exposure of herbicides in farmers have been published from palestine and India [11][12][13][14][15][16][21][22][23][24][25]1,26,3,17,[27][28]. On the other side at highest dose, severe renal toxicity has also been reported after 6-mp treatment. [7] In recent years, the use of such kinds of herbicide has increased. There is a need to study the adverse effects likely to arise from repeated exposure to Penoxsulam. In addition, these experiments are carried out to establish safety criteria concerning to human exposure as many pesticides are toxic for the liver and kidneys. Thyroid gland also plays a significant role in keeping the body functioning normally. But a very few studies have been carried out to see the exposure of herbicide on the thyroid. Therefore, the present study discusses the comparative effects of Penoxsulam herbicide on the liver, kidney and thyroid profile. The present study has been carried out to evaluate the physiological impact of repeated exposure to Penoxsulam herbicide on the Liver, Kidney, Thyroid endocrine profile and biochemical and histological stress in the Wistar rat model.

Animals and Experimental Design
In this study, healthy Wistar rats aged 6-8 weeks, weighing 130-190 gm, and bred at the animal house facility in Shriram Institute for Industrial Research, Delhi, were used. Before commencing the study, permission from IAEC (Institutional animal ethics committee) was taken for this experiment (CPCSEA). Temperature, humidity, light intensity were recorded daily in the experimental room. The room temperature was maintained at 20-24°C with 50-60% relative humidity. The room was ventilated at the rate of approximately 15 air changes per hour with 12 hours of artificial light (8 am -8 pm) each day and the lighting was controlled. Three rats were kept in polypropylene cages (size 430×290×150 mm) with wire mesh tops and each of these cages was tagged with a unique identification number using tail marking. Krishna Valley Pvt. Ltd. provided the standard feeds, and the filtered water was given ad-libitum to the Wistar rats. Feed and water quality was monitored at the NABL-accredited laboratory at Shriram Institute for Industrial Research, Delhi. The floor of the experimental room was swept on a routine basis with a disinfectant solution comprising D-125. The rat cages were rotated every week till the end of the experiment to provide uniform artificial light to the animals. During the experimental period, acclimatization was done for 5 days before administering the doses. Rat cages were tagged with study number, animal number, sex, dose, experiment start date, date of dosing, experiment completion date, and cage number [14].

Grouping of Animals and Dose Level
Animals were randomized according to their body weight and divided into six groups of 10 animals belonging to each sex. All the doses were freshly prepared in a volumetric flask using corn oil as a vehicle. A recovery group was taken to observed the reversibility of the toxic effects. All groups are as follows: (OECD 408, 2018). G1: The control group (only corn oil was administered to the rats) G2: Penoxsulam (Low dose) 100 mg/kg body weight G3: Penoxsulam (Middle dose) 300 mg/kg body weight G4: Penoxsulam (High dose) 500 mg/kg body weight G5: The Recovery control group (only corn oil was administered to the rats)G6: Penoxsulam (Recovery High dose) 500 mg/kg body weight

Acute Oral Exposure Study
In the judgment, the toxic features of a test item is the resolution of the acute oral toxicity in Wistar rats which is usually a step-wise procedure. Hence, this study was carried out to evaluate the acute oral toxicity of Penoxsulam in Wistar rats. A study at the dose of 2000 mg/kg body weight was performed in three nonpregnant female rats as per OECD guideline No. 423. A single oral dose was administered to the animal with the help of a cannula attached to syringes. The animals were fasted overnight before dosing and after 4 hours of post-dosage. In the acute study, animals exposed to Penoxsulam via the oral route did not demonstrate any toxic effect or mortality at the dose level of 2000 mg/kg body weight. Hence, the LD50 range is categorized as per the GHS (Globally Harmonized Classification system) i.e. 2000 mg/kg < LD 50 <5000 mg/kg body weight [15].

Repeated Oral Exposure 90 Days
Based on the acute study and dose range-finding study and existing literature on Penoxsulam, dose levels of 100, 300, 500 mg/kg bodyweight were selected for the main study. The primary study was performed with 60 male and 60 female Wistar rats grouped into 6 (G1, G2, G3, G4, G5, G6). Four groups (i.e G2, G3, G4, and G6) were picked for low dose, middle dose, high dose, and high recovery dose, respectively. They were administered the test compound at dose levels of 100, 300, 500 mg/kg body weight. Similarly, G1 and G5 were classified as control and recovery control groups, and doses were administered to them only through the corn oil vehicle via the oral route with the help of a suitable cannula for 90 days. The purpose of the recovery group is to determine the process of becoming well again after an illness or toxic effect. The animals were observed for toxic signs, haematology and biochemical parameters, body weight, histopathology analysis, and ophthalmology examination. All the rats were anesthetized by CO2 exposure and blood was drawn from the orbital sinus. The whole blood was collected in a gel tube containing K2EDTA anticoagulant, and this blood was studied using AU14091 Beckman Coulter Hematology analyzer. Clinical, biochemical parameters like SGPT (Serum glutamic-pyruvic transaminase u/l), SGOT (Serum glutamine-oxaloacetic transaminase u/l), BUN (Blood Urea Nitrogen mg/dl), UREA, and Creatinine were studied by Beckman Coulter AU480 Clinical chemistry analyser system. The thyroid profile (T3, T4, TSH) was examined by an immunology analyser system COBAS-E-411 ROCHE. At the end of the experiment, the animals were sacrificed and tissues were preserved in 10% neutral buffered formalin for histopathological evaluation (OECD-408).

Statistical Analysis
All data were expressed as mean ± S.D using GraphPad prism software (version 9.2 for windows). The data of body weight gain and clinical chemistry were compared by one-way ANOVA. A 95% confidence level was used to determine statistically significant differences that are dependent on the p-value.

If p value<0.05=significant
If p value>0.05=non significant 3. RESULTS

Clinical Signs
Animal were observed daily for toxic signs, and this study found no mortality or toxic signs and symptoms in any of the dose groups (G1, G2, G3, G5). However, in the high dose (G4) group and recovery high dose (G6) group (at the dose level of 500 mg/kg body weight), all the animals demonstrated ruffled fur, lethargy, anorexia, discoloration of faeces, emaciation, hunched posture, and polyuria. (Table 1).

Mean Body Weight
Body weight gain of the low dose (G2) and middle dose (G3) group animals was comparable to that of control group (G1) animals. However, bodyweight gain in the high dose (G4) group of animals was decreased in males and females when compared to that of the control group (G1) of animals after administration of the dose; though the decrease was statistically significant in the high dose group (G4) (Fig. 1), and body weight gain of the high recovery dose (G6) group animals was comparable to the body weight gain of the recovery control (G5) group animals ( Table  2).

Haematology Evaluation
The hematological blood parameters of all the treated groups (G1, G2, G3, G4) as well as the control group of animals in male and female rats were found to be normal when examined at terminal sacrifice. Similarly, the blood parameters of animals in the recovery high dose (G6) group (500 mg/kg b.wt.) were noticed to be normal (p value>0.05= nonsignificant) to their control counterparts (G5) as the parameters fell within the accepted limits (data not given) in both male and female Wistar rats.

Group & Dose level
Toxic signs and symptoms G1-0 mg/kg body weight No toxic sign and symptoms were noticed G2-100 mg/kg body weight No treatment-related toxic sign and symptoms were noticed G3-300 mg/kg body weight No treatment-related toxic sign and symptoms were noticed G4-500 mg/kg body weight Ruffled fur, lethargy, anorexia, discoloration of faeces, emaciation, hunched posture and polyuria G5-0 mg/kgbody weight No treatment-related toxic sign and symptoms were noticed G6-500 mg/kg body weight Ruffled fur, lethargy, anorexia, discoloration of faeces, emaciation, hunched posture and polyuria.

Biochemical Evaluation
The biochemical parameters examined in the serum sample showed that there was no significant change in the low (G2) and middle dose (G3) group concerning the control (G1) group; However, a slight increase in SGOT (Serum Glutamate Oxaloacetate Transferase), SGPT (Serum Glutamate Pyruvate Transferase), BUN (Blood Urea Nitrogen), Urea, and Creatinine was noticed in animals belonging to the high dose (G4) group that was observed at terminal sacrifice i.e the 91st day, and the parameters of recovery high dose (G6) group animals showed no significant change concerning the recovery control group (G5). Thyroid profile T3, T4, and TSH examined in the serum sample revealed no modifications in any of the dose groups and the control group of animals (Table 3).   The thyroid profile showed no alteration values (p value>0.05=non significant) in any of the treated groups and the control group of male and female rats.

Ophthalmoscopic Examination
Eye examination was done before initiation of the experiment and at the end of the experiment using Ophthalmoscopic equipment in animals belonging to all six groups; no changes were observed in the eyes of both male and female rats during the whole experiment.

Histopathology Evaluation
The liver, kidney, and thyroid were subjected to a histopathology study, and all organs that were examined under the microscope showed the following profiles.

Liver
In control dose (G1) level i.e., 0 mg/kg bodyweight section from most rats showed normal hepatocytes (Fig. 3 a). However, sinusoidal congestion in the liver was observed at a high dose level, i.e. 500 mg/kg body weight (Fig. 3 b) No significant changes in the microscopic liver profile could be noticed at low and middle dose levels, i.e. 100 mg/kg body weight and 300 mg/kg body weight.
After 28 days of post-dosage recovery, the liver was seen in the recovery high dose group of animals compared to their recovery control counterparts.

Kidney
In high dose level (500mg/kg b.wt), shrunken and degenerated glomeruli with loss of bowman's space (FIG. 3 c) occurred in kidneys when compared to kidneys of male and female Wistar rats in the control group showing normal architecture of glomeruli and Bowman's Capsule (FIG. 3 d). No significant changes in the kidney's microscopic profile could be noticed at low and middle dose levels i.e. 100 mg/kg body weight and 300 mg/kg body weight, respectively. In theremaining two groups i.e. recovery control and recovery high dose level, normal cytoarchitecture with a good arrangement was observed in both male and female Wistar rats.

Thyroid
There have not been any alterations in the serum sample of the thyroid profile. A microscopic examination was done, and it did not find any lesions in any of the dose groups and the control group (Fig. 3 e,f).
The histopathological alterations in the present study can be summarized as follows:

DISCUSSION
Researchers utilize animals as replica to help acknowledge and anticipate responses in human beings. Animal representation has delivered the necessary units for exploration and benefit as the origin that has allowed the combustible growing of concern in this area with assembly welfare to both humans and animal species. The utilization of animals in experimental medicine, the science of drugs, drug evolution, well-being safety assessment, and toxicological assessment have become a well-accepted and essential exercise. Animals have been used as a replica for hundreds of years to forecast what chemicals and environmental factors could do to Man [23].
 There are several advantages in using rats; as is evident, humans cannot be used for this research purpose.
Therefore, there is no option of an appropriate (non-rodent) species for pre-clinical toxicology studies based on a contrast of the pharmacokinetics and metabolism of the particular chemical in different laboratory species [10]. The rat species have become a frequent choice because of their metabolic resemblance, small size, relatively willing nature, shortagedness, i.e. 24 to 30 months, and short maturation (gestation). The ample use of rats in research has led to the development of tremendous historical details based on their nutrition, diseases, and general biology. This awareness, along with facts about the species' metabolic reaction and response to toxicants, has proved rats to be a good, known example for predicting human response to foreign compounds. Rats have a short lifespan which is suitable for long-term toxicity studies in which animals need to be exposed for most of their lifetime [19]. Exposure to chemicals has led to increasingly harmful effects on the numerous organs of the body. Within these organs, inadequate responses from cells and tissues is an insult caused by a drug, and its metabolites are determined by the interplay of many factors [21]. The poison acts in small doses, affecting the whole body. Today, it is widely acknowledged that poisonous substances affect the organism by affecting its enzyme system [29].
Due to the accumulation of chemicals in the body, exposure for short periods may not produce immediate effects but repeated exposures can induce delayed effects. Repeated exposure is the adverse or deleterious general toxicological effects that can be both local and systemic. A substance was considered safe in the past if it did not cause immediate death or acute injury to a living organism. Nowadays, a substance is considered relatively harmless if it does not elicit any adverse effects from the biological system, either from organs or single links of the metabolic system [26] Due to the large taxonomic diversity, life cycles vary greatly [24].
On the other hand, the excessive use of herbicides has become a topic of concern as it has finally led to different issues about the ecosystem [25]. Agricultural farmers might be exposed to pesticides while mixing or applying herbicide in thefield [20]. Thus, in every agricultural facility, they must have a thorough training assessment for employees inappropriate work processes and also provide them with the required safety equipment to limit unnecessary exposure because they are not able to understand the instructions or follow label precautions including wearing personal protective equipment (PPE kit) appropriate to the application method. Consumers could be exposed to residues in rice or drinking water, home-lawn applications, contact with treated turf at golf courses and athletic fields, or while swimming in treated lakes. Researchers use animals in their studies to define the potential of a pesticide to cause notorious effects to the internal health of humans [9]. It is relevant to know that these tests are carried out using doses high enough to cause toxicity (poisoning). Effects seen in animals given toxic doses, are unlikely to occur after one-time exposure or low-level exposure in humans. The level of exposure must be considered to estimate the high risks of harmful effects [19].
The unsustainable and unregulated use of pesticides (herbicides) has increased on a daily basis and may cause adverse effects to human beings, including farmers' health. Most farmers who work in such agricultural fields are not aware of these harmful effects. This study has outlined much of the process required for assessing the impact on hormones after chronic exposure to pesticides. There are many known examples of beneficial and potentially dangerous interactions with pesticides resulting from the modern practice of multi-drug therapy in human clinical pharmacology [5]. For close to an era, rats have been used profitably or conveniently in toxicology pre-clinical animal research studies. They will continue to be used for research in the near future.

CONCLUSION
In view of the current study, all biochemical and histopathological alterations correlate with one another. In this study, the data in the high dose group were statistically significant concerning the control group of animals. However, physiological changes were also observed during the experimentation period along with depression of liver and kidney functions after administration of penoxsulam at highest concentration; since no toxic changes has been noticed in any recorded parameters of group-2 and group-3. Hence, it is concluded that the condition of this study had a toxic effect on the liver and kidney at 500mg/kg body weight. Moreover, the herbicide does not imbalances the activity of thyroid hormones (t3, t4, tsh) at the maximum concentration. The findings of this study suggest that the use of Penoxsulam (herbicide) was found to be safe at the lowest concentration.

CONSENT
It is not applicable.

ETHICAL APPROVAL
Institutional Animal Ethics Committee approval was taken before initiation the experiment.