Assessment of profenofos exposure an endocrine disrupting chemical in relation with serum testosterone alterations

The present study was conducted to investigate the toxic effects of endocrine disrupting chemical (EDC) on male reproductive function. Eighty male spray workers were participated and exposed to Organophosphate (Profenofos 500EC) insecticide to evaluate the alterations in serum testosterone level. During study period participants were assessed regarding their exposure to pesticides, protective tools and health conditions. Bloods samples were collected before and after spraying pesticide for the assessment of serum testosterone. Results were analyzed statistically. It was revealed that 70% of the post exposure spray workers had significant decrease in the serum testosterone (4.40±1.25ng/ml P < 0.01) as compared to pre exposure to spray that was 5.80±0.78ng/ml. Out of these 70%, (21%) spray workers were recorded with below normal level of serum testosterone (2.73±0.30ng/ml). Clinical symptoms related to pesticide exposure dizziness, headache, joints pain, nausea, skin and eyes allergy were observed. It was concluded that profenofos may inhibit serum testosterone synthesis by disrupting hypothalamic, pituitarytesticular axis could impair the reproductive function in spray workers.


Introduction
The use of pesticide in agriculture sector has increased throughout the world especially in the developing countries like Pakistan.Pesticides and related agrochemicals are used for controlling noxious pests to increase the yield of crops.On the other hand, indiscriminate use of pesticides is causing severe environmental problems.Controlled use of pesticides is an essential aspect to avoid agrochemical toxicity.
Besides environmental issues, pesticides also cause severe health related problems in living beings by influencing the blood and organs.The adverse effects of pesticides may disrupt the endogenous hormones and endocrine system.Consequently the antiandrogenic effects of various pesticides disrupt the testosterone production in human [1].Exposure to pesticides produces numerous biochemical and physiological alterations in human and their population in the agriculture fields [2,3].Many pesticides are capable to disrupt the synthesis of sex hormones, which can cause reproductive disorders such agrochemicals have been documented as endocrine disrupting chemicals EDCs [4].The farmers/spray workers are directly exposed to pesticides through occupational activities and spraying pesticides on crops.Thus due to mishandling and inefficient safety measures exhibit significant health problems in spray workers by accumulation of pesticide residues in their body.To this, Assessment of serum testosterone alteration on exposure to profenofos in spray workers was the aim of this study.Hence this investigation provides awareness to improve our understanding for reducing the potential risk level to humans.

Materials and Methods Experimental Population and Exposure Assessment
The experimental work was carried in agriculture fields (especially cotton growing areas) from various districts of Sindh, Pakistan.These were particularly pesticide consuming fields where the whole of the year crops are cultivated and harvested.Eighty male spray workers 20 -40 years of age group were voluntarily participated in this study.All spray workers were occupationally related to agro fields.Before exposure spray workers were assessed through a questionnaire regarding their exposure to pesticides, work-related activities, protective measures, general and reproductive health.Scores of protective tools/safety measures and symptoms were recorded for each spray worker.Scoring chart (1-5) was designed for protective tools and exposure related symptoms percentage was also recorded for each spray worker.Later the data of each spray worker was incorporated on Performa.

Test Chemical and Preparation
Profenofos(500 EC) an organophosphate insecticide was used and purchased from authorized dealer of manufacturing company.The test chemical was prepared by following recommended criteria of the Integrated Pest Management (IPM) Sindh Agriculture University, Tando Jam, Pakistan and recommended quantity (Profenofos 500 EC 800 ml /100 liters water) for cotton Sindh Agriculture Department, Govt. of Sindh.

Design and Blood Sampling
Before spraying pesticide, 5ml of blood samples from each spray worker were drawn during 6:00 to 7:00 am.The application of the pesticide on the crops was carried out through lever operate sprayer tank for consecutive three hours.After two to three hours 5ml. of blood samples were collected from all spray workers.Both blood samples before and after spraying labeled accordingly and kept in an ice box.Collected blood samples were brought to the laboratory for the assessment of serum testosterone.

Assessment of Hormones and Techniques
To evaluate the antiandrogenic effect of profenofos, serum testosterone was assessed at research and diagnostic laboratory, Liaquat University of Medical and Health Sciences, Hyderabad, Pakistan.

General Characteristics, Health and Toxicity Parameters
The experimental studies were carried out in the agriculture fields by application of profenofos insecticide through spraying on crops.Mostly the spray workers were uneducated only few attended primary and secondary school education.All spray workers were experienced and occupationally sound and (28%) were found smokers.It was documented that spray workers were used to prepare pesticide dilution without safety measures before application on crops.Furthermore pesticide was sprayed using lever operate sprayers and only a small number of sprayers were in good condition, whereas the others leaked during pesticide spray.Appropriate protective tools / safety measures were not used during spraying activities.Only some spray workers were using a piece of cloth to cover their nose, wearing boots, glasses, and appropriate clothing.The score related to protection and safety measures on pesticide exposure was recorded for all spray workers.The highest score was documented (03) in 15% of spray workers as compared to the maximum score (05).Moreover 40% of participants did not achieve any score for their safety measures (Fig. 1).We also recorded clinical symptoms related to pesticide exposure during and after spraying.Dizziness, headache, backache, joints pain, and nausea after spray tears, skin and eyes irritations blurred of vision, sweating and respiratory disturbances were found common in most of the spray workers (Fig. 2).

Assessment of Hormonal Profile in spray workers
After exposure to pesticide a highly significant (P < 0.01) change in serum testosterone levels were recorded in 70% of the spray workers.From these (70%) with significant change (21%) had below normal serum level of testosterone 2.73±0.30ng/ml(Table 1).This shown disruption in pituitary-testicular axis and exhibit adverse effects on male gonads.After exposure insignificant change (P > 0.05) was also observed in remaining (30%) spray workers.Their after exposure serum testosterone level was 3.67±1.50ng/mlas compared to before exposure 3.73±1.49.Pesticides are major contaminating chemicals in agriculture sector and hazardous to exposed population.These compounds can accumulate in the body's tissues by ingestion.Their bioaccumulation in an organism may lead to a variety of metabolic and reproductive disorders and even outright diseases [18].Results of present study revealed that, farmers were not following appropriate protection and safety measure while spraying pesticides.Hence chemical directly entered in their body and revealed acute poisoning.Our ecological system is contaminated by agro chemicals and indiscriminate usage of agro chemicals and inappropriate spraying tools divulges severe health hazards in farmers [19].Majority of chemical sprayers were not in good condition while spraying on crops in Cameroon.Most of the farmers are not aware of the health hazards caused by the mishandling of pesticides and using inappropriate protective tools [20].The usage of hazardous pesticides associated with inefficient protective tools is detrimental to their health and reproductive Showing number & percentage of spray workers/ farmers (70%) with highly significant change (P<0.01) as mean values (Mean ± SD) of serum testosterone after exposure to profenofos as compared to before exposure & optimal rangeA highly significant change with blow normal serum testosterone level reflected in (21%) spray workers from (70%).Pesticide related clinical symptoms; blurred of vision, nausea, tears, eyes and skin allergies, tiredness, respiratory depression, joints pain and headache frequently occurs in spray workers [33,34].In present study eyes and skin allergy were observed in 50-60% of spray workers.In addition similar types of symptoms with reference to pesticide exposure were recorded during this study.It is concluded that, profenofos may exert adverse effects on reproductive function through inhibition of testosterone synthesis.Moreover it is suggested that, there is an imperative need of awareness to prevent indiscriminate use of pesticides which ultimately minimize the risk level in living beings.

Fig. 1 .
Fig.1.Showing recorded score of protective tools in each spray worker during spraying actvities.The score was calculated by assigning scoring range 1-5 and zero was recoded for no protective tools were used.later percentage of spray workers for each level of score was calculated.

Fig. 2 :
Fig. 2: Showing frequency of symptoms in spray workers exposed to profenofos during experimental studies and on exposure number of reported symptoms among spray workers.

Table 1 :
No. Of Spray workers showing significant changes in serum testosterone directly exposed to Profenofos.Our results are similar in accordance with previous findings; on exposure to profenofos (20%) of the spray workers had below normal testosterone level.This indicated severity of profenofos with drastic effects on the male reproductive function.Environmental toxicants alter the function of male reproductive organs [32].