Effect of a single dose of malathion on spermatogenesis in mice
Eduardo Bustos-Obregn, Patricio Gonzlez-Hormazabal
Biology of Reproduction Unit, Program of Morphology, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile
J Androl 2003
Keywords: testicular toxin; malathion; seminiferous tubule; testosterone
Aim: To observe the acute effect of the organophosphorous insecticide malathion on testicular function in mice. Methods: The effects of a single dose of malathion [240 mg/kg (1/12 LD50)] on plasma acetylcholinesterase (ACE) activity, spermatozoa (epididymal cauda counts and teratozoospermia), testis and plasma testosterone concentration) were evaluated at day 1, 8, 16, 35 and 40 after treatment. Results: The sperm count was decreased significantly 24 h after treatment and teratozoospermia was increased at day 35 and 40. The height of the seminiferous epithelium and the diameter of tubular lumen were decreased at day 8. The percentage of tubular blockade was increased between day 8 and 35. A decrease in testosterone plasma level was observed at day 16 after treatment. Conclusion: Malathion damages male reproduction. The depletion of seminiferous tubules and the increase in teratozoospermia may be a genotoxic damage to the renewing spermatogonia, but the possibility of spermatogenic/spermiogenic disfunction due to a decrease in the plasma testosterone level can not be ruled out.
Malathion is a widely used organophosphorous agropesticide and is categorized as class III (slightly toxic) in terms of risk according to the WHO. Its oral LD50 in rats is 2,100 mg/kg .
The reproductive effects of malathion have not been fully studied. In our laboratory, morphological alterations of testis in mice have been documented after a single dose of commercial malathion . The present work was devoted to a more complete analysis of the male reproductive effects of a single dose of malathion in mice.
2 Materials and methods
2.1 Animals and treatment
Thirty male CF1 mice (10~12 weeks old) were injected i.p. with 240 mg/kg (1/12 LD50)  of commercial malathion (95.2 % w/v in corn oil, Anasac, Santiago, Chile). Groups of 6 animals each were sacrificed at day 1, 8, 16, 35 and 40 post-injection. Six animals injected with the vehicle (corn oil) served as the controls and were sacrificed 40 days post-injection.
2.2 Plasma Acetylcholinesterase (ACE) activity and testosterone level
Heparinized blood samples were obtained through cardiac puncture and the plasma kept at -20 until analysis. The ACE was determined according to Rappaport et al. , using a Sigma Diagnostic colorimetric kit (St. Louis, USA). The testosterone was determined by RIA, using a DPC kit (Los Angeles, USA) with a sensibility of 20 ng/dL and interassay coefficient of variation 11 %. ACE was expressed in Rappaport units and testosterone as mg/mL.
2.3 Testicular histopathology
The testicular histopathology was observed according to Sobarzo and Bustos-Obregn  and Russel et al.. The right testis was fixed in alcoholic Bouin for 8 h, embedded in paraffin, cut and stained with PAS- hematoxyline. Fifty cross sections of the tubules per animal were observed at 1,000 and digitalized to evaluate the seminiferous epithelial height and tubular diameter using a computer program (Scion Image beta 3b, NIH, USA). The percentage of tubules with luminal blockade was determined under a microscope.
2.4 Sperm count and morphology
The sperm count and morphology were assessed as Vigil and Bustos-Obregn . Briefly, the right cauda epididymis was weighed, finely minced and filtered through a piece of gauze. Sperm were counted using a Neubauer hemocytometer and expressed as millions/mg cauda tissue; an aliquot of the epididymal sperm suspension was smeared and stained with hematoxylin and eosin, examined under 1,000 and inspected for head and tail abnormalities (200 sperm per animal).
2.5 Statistical analysis
Data were expressed in median and minimum/maximum values. Differences between groups were determined for the time intervals using the Kruskal-Wallis test and Dunns post-test.
The ACE plasma levels did not show significant changes compared with the controls at all time intervals; The plasma testosterone level was significantly decreased (P<0.01) by day 16 post-injection (Table 1) and the values became normal by days 35 and 40 post-injection, implying subsequent recovery of Leydig cell function.
Table 1. Effect of malathion on plasma acetylcholinesterase activity and testosterone level. cP<0.01, compared with controls.
With the cauda epididymal sperm, there was a marked increase in tail anomalies (P<0.01), but not in head malformations by day 40 post-injection. Sperm counts were not affected at all times post-injection (Table 2).
Table 2. Effect of malathion on sperm morphology and count. cP<0.01, compared with controls.
Table 3 summarizes the histometric observations on testicular tissue. It could be seen that 8 days (one seminiferous epithelial cycle) after injection of the pesticide, both the epithelial height and tubular diameter were significantly decreased (P<0.05), implying atrophy of seminiferous tubules at the particular time interval .
Table 3. Effect of malathion on testicular morphology. bP<0.05, compared with controls.
ACE determination in plasma has been used as a good method to evaluate exposure to organophosphoric pesticides . In the present study, the absence of ACE differences between the treated and controls indicated that a single dose of malathion dose did not elicite general intoxication to the animals, however it caused an early depletion of the seminiferous epithelium with diminution of epithelial height and tubular diameter. Other authors have found a chronic damage after 50 days of treatment . The acute response was similar to that observed with DNA alkylating agents, such as bisulphan  or ionizing radiation . These agents deplete the renewing type A spermatogonia, which respond with proliferation after a couple of weeks , a mechanism also reported when the mitotic regulatory role of the spermatogonial chalones was advanced in our previous study .
However, the seminiferous tubular depletion did not result in significant changes in epididymal sperm counts. If the damage affected type A renewing spermatogonia, the effect should be detected at least 45 days post-injection of malathion (one spermatogenic cycle plus the transport time of sperm to cauda).
The decrease of plasma testosterone levels by day 16 may suggest a toxic effect of malathion on the Leydig cells. Other organophosphoric compounds had been reported to inhibit the non-specific esterase activity in Leydig cells, decreasing testosterone production . Malathion itself had also been reported to lower plasma testosterone levels in rats, without affecting LH levels .
Testosterone, through modulation of P-mod-S in the peritubular cells, could affect Sertoli cell function . The germinal cell sloughing seen in the treated mice may reflect a functional damage of the Sertoli cells.
The sperm tail, but not the head morphology was affected by malathion, which was inconsistant with the results of Giri et al. . The sperm tail morphology could be affected by a number of mutagenic agents .
In conclusion, malathion interferes with mouse testicular function, being toxic both to the somatic (Leydig and Sertoli) and spermatogenic cells (mainly spermatogonia and maturing spermatids). The damage may result from a variety of mechanisms, mainly affecting the DNA structure and function.
The work was partially financed by Banco Santander -Central Hispano, Madrid.
Programme on Chemical Safety Hazardous chemicals in human and environmental
health. The WHO recommended classification of pesticides by hazard and
guidelines to classification. World Health Organization, 2000. Geneve,
to: Dr. Eduardo
Bustos-Obregn, P.O. Box 70061, Santiago 7, Chile.
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