International Journal of Morphology
On-line version ISSN 0717-9502
Int. J. Morphol. vol.25 no.2 Temuco June 2007
Int J. MorphoL, 25(2):245-248, 2007.
Morphometric Analysis of Mice Testicular Tubules after Administration of Malathion and Maca
Análisis Morfométrico de los Túbulos Testiculares del Ratón Después de la Administración de Malation y Maca
Eduardo Bustos-Obregón; Fernando Costa del Río & Luis Sarabia
Universidad de Chile, Facultad de Medicina, Laboratorio Biología de la Reproducción, ICBM, Santiago, Chile.
SUMMARY: Organophosphoric (OP) agropesticidas are amply used to increase food production. However, it has been verified that they induce alterations at testicular level related to the diminution of fertility in humans as in animals. On the other hand, different studies have been made to develop chemical or natural compounds that can induce an antagonistic effect to OP. In previous studies an extract from a plant, from the Peruvian Andes (Maca) has been recognized by its stimulating action on spermatogenesis.
In the present study the effects of both external agents were evaluated on testicular sections of testis of adult male mice on a population of 52 mice CF1, divided at random in 4 groups (Control, Malathion, Maca, Malathion-Maca), with sacrifice intervals of 1,7, 14 and 21 days. By means of morphometric technique and using the "Image Tools 3,1" software, the histology of testicular sections was evaluated, to analyze the degree of alteration induced by these agents. The epithelial height mainly has a rise in day 1, for Malathion group and then fall to day 7 to be normal by day 14. However, Malathion-Maca groups show no changes. The tubular lumen decreases at day 7 and day 14 to be normal by day 21 in Malathion group. However, in Malathion - Maca group, the tubular lumen decreases only at 14 days. The tubular diameter, at day 7 (p<0.01), 14 and 21 in Malathion group decreases respect to control (p<0.05), However, Malathion-Maca groups show normal values. In conclusion, it is possible to establish that the damage induced by Malathion is reverted by 21 days post-administration of Maca.
KEYWORDS: Testis; Morphometry; Mice; Malathion; Maca.
RESUMEN: Los agropesticidas organofosforados (OF) son ampliamente usados para incrementar la producción alimentaría. Sin embargo, se ha demostrado que inducen alteraciones a nivel testicular, relacionadas con la disminución de la fertilidad tanto en humanos como en animales. Por otra parte, diferentes estudios han sido llevados a cabo para desarrollar compuestos químicos o naturales que puedan inducir un efecto antagónico sobre los OF. En estudios anteriores, un extracto de una planta de los Andes peruanos (Maca) ha sido reconocido por su acción estimulante sobre la espermatogénesis.
En el presente trabajo se estudiaron los efectos de ambos agentes externos sobre el testículo. Una población de 52 ratones machos adultos de la cepa CF1 fue dividida al azar en 4 grupos (Control, Malation, Maca, Malation-Maca), con intervalos de sacrificio de 1, 7, 14 y 21 días. Para analizar el grado de alteración inducida por estos agentes, se utilizaron técnicas de histomorfometría con ayuda del programa Image tools 3.1, en secciones testiculares. Nuestros resultados muestran que la altura del epitelio aumentó al día 1 en el grupo tratado con malation, cayendo al día 7 y llegando a valores similares al control, al día 14. Sin embargo, el grupo Malation-Maca no mostró cambios significativos. El lumen tubular, disminuyó al día 7 y 14, para normalizarse al día 21 en el grupo Malation. Sin embargo, en el grupo Malation-Maca el lumen tubular sólo bajó al día 14. El diámetro tubular disminuyó a los días 7 (p < 0.01), 14 y 21 en el grupo Malation, con respecto al control (p< 0.05). Sin embargo, el grupo Malation-Maca mostró valores normales. En conclusión, es posible establecer que el daño inducido por Malation es revertido al día 21 post administración de maca.
PALABRAS CLAVE: Testículo; Morfometría; Ratón; Malation; Maca.
Agropesticides are of amply world-wide use. Among them, organophosphoric (OP) compounds, though restricted in many countries, are however employed in numerous places.
In 1950 the OP Malathion (S-( 1,2-bis-(etoxi-carbonil)-etil)-0,0-dimetil-ditiofosfato) was introduced as being less toxic than Parathion. Both act by inhibiting acetylcholinesterase activity (Hsu et al, 1989). Residues of these OP are environmental contaminants nowadays (Gomero & von Hildebrand, 1990), thus altering the biotic (animals, plants) and abiotic (earth, atmosphere, waters) systems and representing a public health problem in many agricultural communities (Ortiz-Hernández et al., 1997). The OP compounds are lipophilic (Ferrer & Martinez, 1993) and they concentrate in adipose tissue from which they may be released as active metabolites (the "oxon" derivative) and elicit the so-called endogenous re-intoxication (Martinez, 1992).
Biotransformation of OP, mostly by hepatic enzymes, renders them more hidrosoluble to allow excretion of these chemicals (Chapalamadugu & Chaudhry, 1992).
Aside from the inhibition of cholinesterase activity (resulting in accumulation of acetylcholine in the synapses and excessive stimulation of cholinergic receptors in the central and peripheric nervous system, that may even end by death of the subject), OP compounds also damage other tissues. Among them, OP inhibit DNA synthesis in the seminiferous epithelium (Rodríguez & Bustos-Obregón, 2000) and are cytotoxic for spermatogenic cells in mice (Sobarzo & Bustos-Obregón, 2000).
Atef et al (1995) have demonstrated that OP elicit morphophysiological damage of sperm, with cytogenetic damage of male germ cells (Bustos-Obregón & Diaz, 1999; Espinoza et al, 2002).
In the literature there has recently been observations that an extract oí Lepidium meyenii (Maca), administered to rats submitted to an altitude of 4340 meters above sea level (MASL), prevented the spermatogenic damage due to hypoxia (Gonzales et al, 2001). Improvement of male (and female) fertility in rats was also reported by Ponce et al., (2003) using different Maca extracts.
Maca is a Brassicacea cultivated in the Peruvian Andes at altitudes of 3.700 to 4.500 MASL from pre-hispanic times (King, 1987).
The present work evaluates the testicular damage caused by Malathion in mice and the possible protective role of a Maca extract, using a morphometric approach and compares these results with the yield of other evaluative methods (ie. transilumination reported in the literature (Bustos-Obregón et al, 2005).
MATERIAL AND METHOD
Fifty two 3 months old CF1 male mice were used. They were kept under standard animal room conditions, with commercial pellet and water ad-libitum, at 12:12 light/darkness regime and 22°C ± 2 of temperature. Mice were divided in 4 groups, each group with 13 animals (1. M: Malathion; 2. Ma: Maca: 3. M+Ma; 4. C: Control. Each group was subdivided in 4 subgroups according to the days of treatment (1,7, 14 and 21) each subgroup with 3 animals except control group with 4 animals, for administration of NaCl 0,9 % (controls), Malathion (M) (96 % w/v in corn oil, Anasac, Santiago, Chile), single intraperitoneal injection of 80 mg/Kg b.w. (known from previous experiments to be testicular toxicant in rodents) (Contreras & Bustos-Obregón, 1999). Maca (Ma) was administered daily by intubation of the esophagus of the animals at a dose of 66,6 mg/day (Maca extract was prepared as indicated in Bustos-Obregón et al, 2005).
At the end of each period, mice were anesthesized with ether and then sacrificed by cervical dislocation. Testes were dissected out and fixed in Bouin's solution to be processed by routine histological techniques. Parafin frontal sections (5 \im) of the equatorial part of the testis were obtained. One section out of every 10 were mounted and stained with PAS (Peryodic Acid Schiff) and hematoxylin and inspected under 1000X in a Nikon microscope. For each group, 200 tubules were evaluated after transferring the digitalized imagen to a PC, using a TV high resolution camera attached to the microscope. Images were analyzed for tubular diameter, and lumen, epithelial height and percentage of area of interstitial space, using the program Image Tools, 3.1 as a software (http://ddedx.uthscsa.edu/dig/itdesc.html).
Statistical analysis of data was done using the Anova test and non parametric test of Kruskal-Wallis followed by the Dunn multiple comparison test.
Epithelial height (Table I): there is an increase in the M group, in agreement with the changes that the OP elicits starting from day 1 and a decrease at day 7 to be back to normal values at day 14. Results for Ma show an increase at day 14 and normal values at day 21. However, M+Ma group showed an increase only at day 14 in epithelial height, and no effect at days 1 and 7 suggesting a reversion of effect of M at day 1 and 7.
Concerning luminal mensuration (Table II) a clear tendency to diminish luminal diameter is seen at day 7 in the group treated with Malathion (p< 0.05), probably due to the depletion of late spermatids, as aresult of acute to xicity of M. The M+Ma group does not show a significative variation at these same days.
The tubular diameter (Table III) shows a decreased at days 7, 14 and 21 in M group. However, M+Ma group does not show variations, suggesting a reversion of the effect of M at days 7, 14 and 21.
Studies on the toxic effects of OP are numerous, dealing with different topics such as geno- and cytotoxic effects, including abnormal protein assembling and DNA alterations (De la Cruz, 2004). The testis is one of the organs afected by OP. Since spermatogenesis is a complex process, encompassing cell proliferation and differentiation, cell damage and mechanisms of toxicity are difficult to establish (Bustos-Obregón, 2001). Complete spermatogenesis in mice takes 33,2 days (Oakberg, 1958) so that more than one cycle (8,3 days) has to be analyzed after a single exposure to a testicular toxicant to gain insight into the damage of the seminiferous apithelium. M interferes with the normal spermatogenic cycle in mice (Contreras & Bustos-Obregón) after a single dose and at intervals from 4 to 26 days; resulting in increased teratozoospermia and acrosomal and flagellar damage.
Damage is also noticed in immature mice exposed to Parathion (Sobarzo & Bustos-Obregón), with reduced tubular diameter at days 7,14 and 21, and a decreased epithelial height (noticed in adults after M treatment only at early intervals, ie 1 and 7 days), and a clear recuperation effect in M+Ma, in concordance with reports of Gonzales etal. (2001 and 2003) in rats. It seems that the sensitiveness of the seminiferous epithelium changes with age of animals and that the post-meiotic germ cells are more sensitive to the OP. Ma helped recovery of mitotic activity in mice treated with M (Bustos-Obregón et al, 2003), in agreement with our present histological observations that in the M+Ma group, atrophy and vacuolization of the epithelium decreases in this group compared to M.
According to Wing & Christensen (1982) morphometric variations of the seminiferous epithelium depend on the stage of the cycle of a given tubular section, a fact that is supported by our observations.
If morphometry is not done in correlation with staging of the tubules, only gross considerations can be assumed.
In general, under our conditions, it can be concluded that morphometric differences between controls and the experimental groups are seen from day 7 onwards and values tend to revert to normal after 21 days of observation.
The different techniques to analyze spermatogenic damage and morphometry (staging: Wing & Christensen; morphometry: Sobarzo & Bustos-Obregón; histopathology and associated cytochemical, radioautographical and immunocytochemical techniques: Bustos-Obregón & González-Hormazábal, 2003; transillumination: Bustos-Obregón et al., 2005) are all in wide use and give complementary results.
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Prof. Dr. Eduardo Bustos-Obregon
Received: 21-12-2006 Accepted: 03-03-2007