Perinatal effects of scorpion venoms: maternal and offspring development

Scorpion envenomation is a public health problem, especially in tropical and subtropical countries. Considering the high incidence of scorpionism in some areas, pregnant women and nursing mothers may be possible victims. Scorpion stings alter the release of neurotransmitters and some cytokines. These mediators act as organizers and programmers in the adequate formation of the nerves, and non-physiological concentrations of them during the brain organization originate disorders and diseases that can appear later in the life of the individual. Despite the importance of this subject, there are only a few studies showing the effects of scorpion venom on maternal reproductive development, in the morphology and physical and behavioral development of offspring. The present review article summarizes the major findings on this issue. Biochemical changes in the blood – such as hyperglycemia, increase on the level of sodium and on the creatinine concentration – are observed after scorpion sting in humans and experimental animals. Some studies in the literature demonstrate that the scorpion venom affects the maternal reproductive development in humans and in experimental animals, increasing the frequency and amplitude of uterine contraction and the number of resorptions. The venom can also lead to some alterations in the embryonic or fetal development increasing the total weight of fetuses and of some organs. Moreover, it affects the general activity and locomotion during childhood and adulthood, and the anxiety level in adult females and males. It also alters the number of hippocampal neurons and interferes in the level of some cytokines. Altogether, it is evident that the venom, when administered during the pregnancy or lactation, affects the development of the offspring. Studies are being conducted to determine the actual participation of the venom in the development of the offspring, and to what extent they are detrimental to animal development.


Background
Scorpion envenomation is a public health issue in many places around the world, especially in tropical and subtropical countries, not only due to the high incidence of accidents, but also because the venom can induce morbidity in stung patients [1]. On account of the high incidence of scorpionism, pregnant women and nursing mothers may be possible victims of scorpion envenomation.
According to Isbister [2], despite the great number of scorpion accidents per year, most cases are minor, with localized pain and minimal systemic involvement. Pain is present in more than 95% of the envenomation cases whereas systemic manifestations are less frequent [3,4].
Due to the variety of scorpions found worldwide, a range of clinical cases may occur [3]. Although some differences are observed, systemic envenomation is characterized by relatively similar neurotoxic excitation syndromes that result from the release of neurotransmitters and due to the action of neurotoxins on sodium channels [2][3][4]. Besides the release of neurotransmitters from the autonomic nervous system, scorpion venom may also provoke changes in the central nervous system, including epileptiform activity and neuronal damage in hippocampal neurons [5][6][7][8][9][10]. It is also responsible for tissue alterations, which lead to the release of inflammatory mediators such as cytokines [11][12][13].
There are approximately 1,500 species of scorpions described worldwide and only about 30 of them belong to the Buthidae family, which is considered dangerous for humans and responsible for serious cases of envenomation and even death [7]. Some physiological alterations that occur during the envenomation caused by these scorpions can impair the proper development of the pregnancy and the fetus. For example, the maternal adequate oxygenation is of utmost importance to the fetus, as well the adequate level of glucose in the blood or the blood pressure [14].
Neurotransmitters and cytokines regulate essential processes of life. In addition, during the pre-and postnatal development, they act as organizers and programmers in the adequate formation of the nervous system [15]. Non-physiological concentrations of these mediators during the brain organization originate disorders and diseases that appear later in the life of the individual, which means they can act as endogenous teratogens [15].
During the development of the nervous system, neurotransmitters perform important roles [16]. The neuronal development is characterized by a combined action of trophic and degenerative processes leading to a complex organization of the mature central nervous system. This is a selection process in which certain neurons are stimulated to survive, while others are suppressed or even eliminated. Some evidence suggests that classical neurotransmitters are actively involved in these processes as promoters or suppressors of neurons [17]. Monoamines appear early in embryos, before the differentiation of neurons, and several studies have demonstrated that they are fundamental in the regulation of the brain development before assuming their role as neurotransmitters in the mature brain [18,19]. Cytokines perform an important role in the development, proliferation, survival, differentiation, and growth, as well as in the neuronal regulation of neuronal synapses [20].
Few reports are documented in details in the literature regarding the effects of the scorpion venom in the perinatal phase, and the available data are somewhat controversial. Some authors state that a scorpion sting during pregnancy does not affect the mother or the fetus. In this sense, Langley [21], in an extensive review of studies published between 1966 and 2002, showed that no adverse consequence was found in human mothersor their fetuseswhen they were stung by a scorpion during the pregnancy. Similarly, Kaplanoglu and Helvaci [22] described the clinical findings of scorpion stings in 11 pregnant women. All patients developed mild envenomation and pregnancy complications were not observed. In a clinical case described by Bozkurt et al. [23], an early-stage pregnant woman stung by a yellow scorpion had excruciating pain without any other local or systemic complaints, and there were no consequences for the mother or the fetus. Ismail et al. [24] reported 52 cases of scorpionism, among those, two were pregnant women who presented no further consequences for them or their fetuses.
Although a retrospective study carried out in Tunisia from 1990 to 2004 with twenty pregnant women demonstrated neither maternal or fetal death, nor preterm fetal delivery, it reported two patients who manifested intense pelvic pain and one of them had vaginal bleeding [25]. Moreover, Ismail et al. [26] reported abortions in pregnant women whereas Zengrin et al. [27] observed seizure and eclampsia in a late-stage pregnant woman stung by a scorpion in Turkey. Kankonkar et al. [28] noted that a number of pregnant women died due to the red scorpion sting in India, but no details were provided. Finally, Leibenson et al. [29] reported a case of a pregnant woman that gave birth to a stillborn infant following a yellow scorpion sting.
Despite the importance of the subject, there are only a few studies showing the effects of the scorpion venom on the maternal reproductive development and on the morphology of the offspring, as well as on their physical and behavioral development. This review summarizes the major findings on this issue, mainly regarding the experimental studies. According to Brown et al. [14], the evidence regarding the maternal history and the treatment of scorpion envenomation during pregnancy is derived from animal data, and extrapolations to humans are limited due to differences in the study design and interspecies differences and responses to the envenomation.

Hematologic changes in pregnancy
Throughout the course of the pregnancy until birth, the biological systems of females are deeply changed in order to ensure a good fetal development [30]. Any disorder may threaten both the mother and the child. Hyperglycemia and maternal hypertension, for example, generally contribute to embryonic and fetal malformations, as well as teratogenicity and fetal lethality [31].
Scorpion venom induces a body system response that leads to hyper-or hypotension, hypothermia, tachycardia, leukocytosis, hyperglycemia, myocarditis, pancreatitis, respiratory disorders and other complications, both in humans and in experimental animals [32]. Biochemical and hematological changes, as previously stated, such as hyperglycemia, hyperamylasemia, increase in serum activities of creatine kinase and aspartate aminotransferase and increase in hematocrits, red blood cells count, and in hemoglobin concentration have been observed after the scorpion stings in humans and experimental animals [4,[32][33][34].
Maternal blood alterations including hyperglycemia, increase in sodium and creatinine concentration were observed [35]. Ben Nasr et al. [31] showed that the Buthus occitanus tunetanus venom causes biological changes in the plasma of pregnant females, such as alterations in blood pressure and in the lipid peroxidation levels, and increase in the concentration of creatinine, βestradiol and progesterone. Two pregnant women accidentally stung by scorpions presented hypertension associated with pelvic pain and vaginal bleeding. Neither maternal or fetal death nor preterm fetal delivery was observed [25]. Another reported case showed that a pregnant woman stung by a scorpion had discreet biochemical and physiological changes; however, during the period of observation, she was diagnosed with eclampsia [27]. The experimental injection of B. o. tunetanus venom in pregnant rats caused similar effects on maternal blood parameters, including increase in sodium level and reduction in glucose level, increase in the concentration of creatinine, urea and white blood cells [36]. These observations suggest that the envenomation may cause disturbances in the gestational process and in the development and well-being of the fetus [31].

Alterations in the maternal reproductive system during pregnancy
Some studies suggest that the scorpion venom provokes alterations in the maternal reproductive system in humans and in experimental animals [13,25,31,[37][38][39].
It was shown that the Leiurus quinquestriatus venom increases the frequency and amplitude of uterine contractions mediated by the release of kinins [40]. Ismail et al. [26] showed that the Androctonus amoreuxi venom, injected repeatedly from the 5 th to the 8 th day of pregnancy, increases the number of fetal resorption and causes defects in ossification and weight loss in survivor pups. Moreover, Buthus minax venom causes skeletal malformations in goats and induces abortions in pregnant women [26]. Buthus occitanus venom contains a peptide that enhances bradykinin levels leading to muscle contraction in isolated uterus of rats, whereas T1 toxin from Tityus serrulatus venom causes the same effect [41,42].
L. quinquestriatus venom produces a marked effect on the frequency and amplitude of uterine contractions of pregnant rats that seem to be more sensitive to the effects of the venom at the beginning of the pregnancy [43].
A study carried out with T. serrulatus venom demonstrated that a single injection on the 5 th or 10 th day of gestation in rats did not cause any significant change in the maternal reproductive system. However, two dams treated on the fifth day of gestation presented preimplantation loss and other two had post-implantation loss without changes to maternal weight gain [37]. The same venom at a higher dose injected into females on the 10 th day of gestation increased the number of postimplantation losses [38]. Likewise, a single dose injection of Tityus bahiensis venom in pregnant rats on the 5 th or 10 th gestational day caused an increase in the number of pre-implantation losses of blastocysts [44].
Some studies demonstrated that scorpion venoms affect cytokines, which may be involved in the observed pregnancy losses [12,33,45]. Cytokines are important mediators in the gestational process, which lead to a successful pregnancy since they are involved in the implantation of the blastocyst, embryo formation and development, and especially in the development of the central nervous system [46]. The uterus promotes an immune adaptation during pregnancy to prevent maternal rejection [47]. The IL-10 cytokine seems to be the key to maintaining the gestational process due to this protection effect of the fetal-placental unit, inhibiting the secretion of inflammatory cytokines, such as IL-6 and INF-γ [48]. An interference of the venom with these mediators would be the cause of the alterations observed after the envenomation.

Alterations in offspring morphology
In addition to affecting the appropriate development of pregnancy, scorpion venoms can also lead to some alterations in the embryonic or fetal development. T. bahiensis venom, when administered to rats on the 5 th or 10 th gestational day, caused changes in the offspring, such as increase in the fetus, placenta, lung, liver and heart weight, although the visceral macroscopic analysis demonstrated a normal appearance of the organs. The skeletal analysis of these pups showed no structural alteration [44]. Similarly, the venom of the T. serrulatus provoked an increase in the weight of the lungs, liver and placenta of fetuses of mothers who received a single dose of this venom during pregnancy [37]. Ben Nars et al. [49] showed a similar increase after the injection of the B. o. tunetanus venom. According to these authors, such effects are related to disabled placental function, maternal hypertension and metabolic disorders.
The augmented weight of pups is not traditionally considered fetal damage, but the increased weight of the liver, lungs and heart can be considered a consequence of the envenomation. Changes in the weight of pups, placenta and of some organs can be explained by alterations in the placental and embryonic pathway. Among the numerous metabolic processes of the placenta, many are considered essential for a good embryonic development. For the fetuses, the placenta is an all-in-one endocrine organ, that is, a combination of the gastrointestinal tract, kidneys, liver, lungs, spleen, and thymus. It carries on nutrients to the fetal circulation and is responsible for the metabolism of this system [50,51]. An increase in the weight of the placenta could indicate changes to the metabolism of these nutrients that would be not necessarily a good or a bad thing for the pups [37]. The increase in fetal weight could be a consequence of an increase in the weight of the organs, or alternatively, an increase in the muscular mass or body fat of these pups [44].
The respiratory system appears to be particularly sensitive to the venom [52]. During envenomation, the lungs accumulate large quantities of scorpion venom [53]. The increase in pulmonary weight of the offspring could confirm the sensitivity of this organ to the venom. The mechanism by which the venom increases the weight of the lung in the offspring is not clear, but lung development can be changed in various ways, including by the release of glucocorticoids after the contact with the venom [54]. Cytokines appear to be clearly involved in the alterations found in the venom-treated offspring. Variations in the levels of IL-1 could be responsible for the increased weight of the placentas, since the IL-1 system is associated with the placental damage and increase in the brain [55].

Effects of envenomation during pregnancy on postnatal development
Physical and reflexological development From birth to senescence, humans and animals develop a set of motor skills that ensure their survival and physical independence. Since pregnancy is a delicate phase in the formation of several biological systems, external or internal agents could generate fetal development disorders, damaging structures responsible for the physical, motor and behavioral development of the offspring.
Lactation is another important phase for the complete development of the offspring, during which the largest outbreak of brain growth and maturation of various organs and biological systems occur. The blood-brain barrier and the blood-gut barrier are still immature and depend on nutrients provided through breastfeeding for proper development [56,57]. Some experimental studies have demonstrated that the injection of scorpion venom during pregnancy or lactation can induce alterations in the postnatal development. The injection of the T. serrulatus venom on the 10 th or 16 th gestational day causes an increase in the weight of the pups at birth, and alterations of some physical parameters including delay in ear opening and palmar grasp, and advance in the ear unfolding, and subtle changes in the motor reflexes [38]. T. bahiensis venom, experimentally administered on the same days, caused similar alterations, but with gender differences [58].
In another study, when T. bahiensis venom was injected into mothers during lactation on the 2 nd , 10 th or 16 th days, subtle alterations were observed in the physical parameters of the pups [59]. Such variations included delay in ear opening, in the incisor tooth eruption and unfolding of the ear, as well as alterations in the postural reflexes, such as delay in the surface righting and negative geotaxis [59].
According to Martins et al. [59], the venom interferes, directly or indirectly, with the maturation of the systems or alters some processes, such as cellular differentiation or repair. The venom promotes the release of several inflammatory mediators, among them, IL-6 [11]. It has already been demonstrated that prenatal administration of IL-6 increases the birth weight, since it reaches the fetus through the placenta [60,61].

Behavioral development
The evolution of the locomotion, from pregnancy until adolescence, does not involve a single system, but it depends on a variety of systems, such as the sensory, motor, and postural systems and neurotransmitters [62]. During the long period of neural maturation there are critical periods that are subject to internal and external influences, inducing various behavioral effects [63]. Scorpion venom can be included among the external factors that lead to behavioral changes in the offspring.
In this sense, it was demonstrated that the administration in rats of T. bahiensis venom on 16 th day of pregnancy increases the general activity and locomotion in the male offspring during adulthood, and locomotion in pups [58]. On the other hand, the injection of the same venom on the 10 th day of pregnancy decreases the general and locomotor activities of males during adulthood [39]. During lactation, depending on the time of application of the venom, there is a subtle increase or decrease of this behavior, in both males and females, in childhood and adulthood [59].
According to Vinay et al. [64], in rats, the peak of the motor development takes place around the second week of life. The direct or indirect action of the venom would be responsible for injuries in the formation of motor pathways or, alternatively, for an unbalance in the release of important neurotransmitters, such as dopamine, which is critical for the control of the motor activity [63,65]. Females are more susceptible to prenatal stress [66]. Maternal proinflammatory cytokines are associated to fetal cerebral injuries [67][68][69]. Immune reactions mediated by cytokines during the embryonic period or during childhood can increase the risk of cognitive and/or psychobehavioral mismatches [70]. The administration of T. bahiensis venom during pregnancy decreases the level of anxiety in males during adulthood, while in females it is increased [39]. During lactation, the anxiety level in females is decreased, but alterations in emotionality were observed in both genders [59].
According to Mathews [71], anxiety and memory are intrinsically related, and anxiety is necessary for the memory formation. In the case of the venom, it is possible that emotionality and cognition are affected by different routes within the nervous system, since there is no evidence of any damage to the formation and consolidation of the memory.

Structural alterations to the hippocampus
The hippocampus has an important participation in the emotional cognitive control and in motivational processes [72]. Due to its evident importance, histological studies were conducted in order to evaluate whether this structure is affected by the venom, and whether there is any correlation with the physical, reflexological and behavioral alterations caused by the venom [39,59].
T. bahiensis venom, when administered to pregnant rats, causes neuronal loss in the CA1, CA3 and CA4 hippocampal areas of the offspring [39]. On the other hand, when applied during lactation, an increase in the number of hippocampal neurons in pups and in adults was observed [59]. Several studies indicate that prenatal stress causes a significant reduction in the cellular proliferation; however, depending on the intensity of the stress, this neurogenesis can be suppressed or reinforced in specific cerebral areas [73,74]. During the perinatal phase, an exponential growth of the brain takes place. More than 85% of the hippocampal neurons are formed during this period and, at the end of the first week, a physiological death of approximately 70% of the neurons occurs [75]. The peripheral activation of T cells is associated with an increase in the cellular proliferation and hippocampal neurogenesis [76]. The offspring of mothers treated with the venom during pregnancy or lactation had an increase on the level of cytokines [13,59].

Cytokines
It was demonstrated that T. bahiensis venom injection during pregnancy decreases the level of INF-γ, IL-10 and TNF-α in the fetuses 24 h after the application, and it increases the level of IL-1α six hours after the application [13]. When the injection occurs during the lactation period, there is an increase in the cerebral level of INF-γ in pups 24 h after the maternal envenomation [59].
During pregnancy, cytokines take part on the blastocyst implantation, formation and development of the embryo [46]. During the perinatal period, cytokines participate in the development of the central nervous system, and are extremely important for the neurogenesis, apoptosis and control of neuronal processes [77]. According to Lin et al. [48], IL-10 is responsible for maintaining the pregnancy and for protecting the placenta-fetal unit. The implantation losses observed after envenomation could be explained by a reduction on the level of IL-10 [38,44].
The activation of the immune system, particularly IFN-γ, is associated with neurogenesis [78][79][80]. High levels of proinflammatory cytokines generated by the mother or the fetal immune system are associated with abnormal fetal brain development and neurological disorders that may be responsible for unexplained behaviors occurring in adulthood [81].
From the gestation until approximately the second week of life, the development presents several windows in the maturation processes that can suffer damages by internal and external factors [82]. The changes induced by the venom on cytokine levels during gestation or lactation could be related to the alterations in the physical, cognitive and behavioral development of the offspring [39,58,59].

Conclusions
Altogether, it is evident that scorpion venom, when administered during pregnancy or lactation in animal models, affects the development of the offspring. However, further studies in this area are necessary in order to determine the actual participation of the venom in the development of the offspring, and to what extent it is detrimental to the animal development. Moreover, the mechanisms of action and the systems affected by scorpion venoms during the perinatal phase should be the subject of further studies, as well as the pharmacokinetics during pregnancy.