Exposure to neurotoxic chemicals during the developmental stages is a source of concern to the health and well-being of the developing organisms owing to the vulnerability of the central nervous system (CNS) during this period coupled with the immaturity of the brain-blood barrier29. The present study revealed alterations in the various developmental parameters in F1 male rats following gestational and lactational exposure to CP and CY.
The litter size is an important indicator of developmental and reproductive failure or success. In the present study, the mean litter size significantly decreased in the group exposed to the insecticide mixture compared to that of the other groups. This agrees with findings from previous studies on CP30,31 and CY32 in rats. The decrease in litter size has been documented as one of the foetotoxic signs of pesticide exposure and is attributed to several factors, including the ability of CP and CY to cross the placenta barrier33,34. Besides, previous studies have shown that CP30 and CY32 promote pre-implantation losses, which may affect litter size. Furthermore, oxidative injury to the fallopian tubes35 by the pesticide mixture may have created an unfavourable medium for implantation of the blastocysts, thereby resulting in lower litter size36. Increased apoptotic damage to the embryo observed with exposure to certain pesticides, including CP during the pre-implantation and post-implantation period37 may have contributed to the decrease in litter size in the group exposed to the pesticide mixture only. However, pre-and post-treatment with ML improved the litter size in the F1 male rats exposed to the pesticide mixture, partly due to its antioxidant properties. Melatonin acts as a direct ROS scavenger38 and as an indirect antioxidant by stimulating the synthesis and release of antioxidant enzymes39.
The present study has also revealed a significant decrease in the viability of pups from dams co-exposed to CP and CY, as a greater number (22%) of the pups in the CC group died. Cypermethrin has been found to induce DNA damage40, chromosomal aberrations41 and steroid hormone disruptions42. Indeed, Madu43 demonstrated a decrease in the number of live-born fetuses following CY exposure. Similarly, CP has also been shown to cause genotoxic effects through DNA damage and cell apoptosis40,44. The improvement in live/dead ratio following pretreatment or posttreatment with ML suggests that the antioxidant agent counteracted the pesticide-induced toxicity possibly by protecting against ROS-induced DNA damage and protein oxidation45 coupled with its ability to decrease the level of certain pro-apoptotic enzymes like caspases 3 and 946 and its ability to protects against genotoxic damages47, which eventually reduced prenatal and neonatal mortality.
The litter weight has been widely used as an indicator of foetotoxicity of a test substance48. The result of the present study showed a decrease in the litter weight of the F1 male generation of the CC group throughout the lactation period, relative to that of the other groups. This result agreed with previous findings following CP30 and CY43 exposure. Perera et al.13 reported an association between CP exposure and low-birthweight among the African-American population, while a similar relationship has been between umbilical cord plasma CP levels and foetal birth weight. The relative decrease in the pattern of weight changes in the groups co-exposed to the two pesticides may be partly due to cholinergic and oxidative stress, engendered by the pesticides49,50.
Pre-treatment and post-treatment with ML showed a significant increase in litter weight relative to that of the CC group right from PND 0 to PND 21. Apart from expressing ML receptors in the placenta, melatonin has been shown to protect against oxidative damage induced in the rat placenta. Maternal treatment with ML in the present study may have improved placental efficiency, which therefore aids in the restoration of litter weight, partly due to an increase in the expression of placental Mn-SOD and catalase by the up-regulation of the placental antioxidant enzymes51. Furthermore, ML has been shown to improve blood and nutrient supply to developing foetus through improvement in uterine blood perfusion52.
The crown-rump length (CRL) is a measure of fetal growth rate and has been used to evaluate growth retardation in response to the intrauterine exposure of the foetus to a noxious environmental chemical substance53. The present study showed a decrease in the mean CRL of F1 male generation rats in a group co-exposed to the pesticide mixture, indicating decreased foetal growth rate. This finding agrees with previous reports following gestational exposure to CP30,31 and CY53. The growth retardation in the newborn in the present study may be due to the ability of CP to concentrate in the milk. A concentrated form of CP residue has been demonstrated in the breast milk of mothers54 exposed to the pesticide, as it interacts with milk protein55, thereby posing a lot of danger to the newborn. Similarly, OP compounds also alter the activity of the milk lipase enzyme, resulting in diminished secretory function of the mammary gland, resulting in interference with the nursing of the offsprings56,57. In addition, CP easily crosses the placenta barrier58 causing direct cytotoxicity to the developing foetuses, thus impairing their growth and well-being. Furthermore, the pesticide mixture’s ability to induce oxidative stress and other forms of stress may have created an unfavourable uterine environment for foetal growth and development, culminating in a reduction in CRL.
Pre-treatment and post-treatment with ML in the present study caused a significant increase in CRL, indicating an improvement in the foetal growth and a decrease in foetotoxicity, apparently due to its antioxidant property. Melatonin, up-regulates the activity of various antioxidant enzymes, while also enhancing the action of other antioxidants, such as ascorbate and tocopherol59. Through its mitigation of oxidative and cholinergic stress60,61, ML may have provided a better intrauterine environment necessary for foetal growth, in addition to reducing cytotoxicity induced by the pesticide mixture.
Anogenital distance (AGD), which has been used to gauge reproductive toxicities is a sexually dimorphic measure of genital development and a marker of endocrine disruption in animals and humans62. The AGD is dependent on prenatal exposure to androgens, which stimulate the growth of the perineum63. Although not significant, AGD in the F1 generation of male rats exposed to the pesticide mixture in the present study was relatively shorter than any of the other groups, including groups treated with ML. This result suggests that the two pesticides have some degree of anti-androgenic effect on the foetuses, in agreement with findings from a previous study that reported low AGD following CP exposure30. Similarly, CY exerted anti-androgenic effects in androgen receptor gene assays64.
The restoration of the insecticide-induced deficit in AGD of F1 male rats in ML pre- and post-treated groups suggests its ability to ameliorate this developmental disorder. This could be partly attributed to the ability of ML to protect the Leydig cells of the developing foetus from insecticide-induced oxidative damage65, thereby retaining its capacity to produce testosterone during the stage critical to foetal urogenital development.
The present study recorded a slight delay in the time of the ear opening. This agrees with the finding of previous studies following gestational exposure to CP66 and CY67 in rats. Stimuli from the skeletal muscles, which have been reported to play a role in the foetal development of the external ear, may have also been partly responsible for the delay in the time of opening of the ear in the present study. Melatonin was able to normalise the time of pinna opening, possibly by reducing both the maternal and foetal toxicity, engendered by the pesticides possibly by its antioxidant properties.
The significant delay in the time of eye-opening in F1 generation from dams in the CC group indicates that the insecticide combination impaired this important developmental landmark. Several studies have demonstrated the ability of CY to cause delay of eye-opening in rats67,68. The delay in eye-opening may be partly due to retarded synaptogenesis of the primary visual cortex (VI)69, possibly due to oxidative stress provoked by the insecticide mixture. Oxidative stress plays an important role in synaptogenesis through the activation of mitogen protein activated kinase (MAPK) signalling pathways70.
Pretreatment with melatonin was able to reduce the time of eye-opening, possibly due to the mitigation of oxidative stress evoked by the insecticide mixture, which allows the normal process of synaptogenesis of the primary visual cortex (VI). Melatonin may also have reduced the activation of the MAPK signalling pathway since antioxidants have been shown to reduce the activation of p38 MAPK71. The implication of improved synaptogenesis by melatonin possibly due to the reduction of oxidative stress is early maturation of the visual cortex, which resulted in the reduction in the time of eye-opening.
Undescended testicles are the most common congenital birth defect in male children and were generally accepted to affect 2-4% of baby boys72. In the present study, there was an increase in the time of testicular descent in the group exposed to pesticide mixture only, which indicates changes in the physical parameter of sexual maturation73. Testicular descent is testosterone dependent, hence a decrease in testosterone concentration, which has been documented to be engendered by CP74 and CY75 may have been partly responsible for the infraction on this developmental parameter. The improvement in the time of testicular descent in the FI generation from dams pretreated with melatonin may be due to its widely proven antioxidant effect, which may have protected vital reproductive endocrine organs/cell such as the hypothalamus, pituitary gland and the Leydig cells, thus, allowing them to regulate the synthesis and secretion of androgens. A previous study has shown the ability of ML to mitigate CP-evoked disruption of the pituitary-gonadal axis76.
Although the present study did not evaluate the redox status of the animals under observation, it is however known that exposure to pesticides causes genetic and epigenetic modifications, endocrine disruption, mitochondrial dysfunction and oxidative stress77. Reactive oxygen and nitrogen species play some roles in regulating essential cellular signalling pathways such as cell differentiation, proliferation, migration and apoptosis78. This may have been partly responsible for the developmental toxicity caused by the pesticide mixture in the present study. The mitigation by melatonin may also have been partly due to its antioxidant effect through direct and indirect pathways. The direct antioxidant and free radical scavenging properties of melatonin are mainly due to its electron-rich aromatic indole ring, which makes it a potent electron donor that can significantly reduce oxidative stress79,80. Indirectly, melatonin does activate melatonin (MT) 1 and MT2 receptors and upregulate antioxidative defensive systems by increasing the expression or activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase81. Apart from its antioxidant effect, the ability of melatonin to mitigate the toxic effect arising from in utero exposure to mixture CP and CY in F1 generation may be due to its antiapoptotic effect. Melatonin has been shown to modulate the Bcl-2 protein expression, blocks Bax proapoptotic activity via the SIRT1/NF-kB axis with a consequent and significant inhibition of Cytochrome C release and the lack of apoptosome formation and caspase 3 activations82,83.
In conclusion, the present study has demonstrated that gestational and lactational co-exposure to CP and CY alter some developmental landmarks in the resulting male F1 generation, which may adversely affect their future developmental and reproductive potentials. Melatonin, when given before gestational and lactational exposure to insecticides or even after their exposure, acting both as a prophylactic and curative agent mitigated the developmental alterations in the F1 generation.