Prolonged testosterone 17ß-cyclopentylpropionate exposition induces behavioral, ovarian, oviductal, uterine and reproductive disturbances in female mice.

Anabolic-androgenic steroids (AAS) abuse is often associated with metabolic disorders and infertility. However, the current evidence on AAS-induced reproductive toxicity is mainly based on male studies. Thus, AAS repercussions on female reproductive capacity remain poorly understood, despite scarce evidence that fertility determinants may be more severely impaired in females than males exposed to these drugs. Accordingly, this study used an integrated framework to investigate the impact of different testosterone 17ß-cyclopentylpropionate (TC) doses on pain sensitivity, aggressiveness, anxiety, sexual behavior, ovarian, oviductal, uterine and reproductive morphofunctional and molecular outcomes. These parameters were used to explore the reproductive capacity in female mice exposed to this synthetic testosterone ester. The animals were untreated or intraperitoneally treated with 5, 10 and 20 mg/kg TC every 48 h for 12 weeks. Our findings indicated that testosterone was upregulated while the hormones luteinizing, follicle-stimulating, estrogen and progesterone were down-regulated by TC. This AAS also exerted deleterious effects on anxiety, aggressivity, nociception, exploratory and sexual behavior in female mice. Concurrently, TC attenuated ovarian follicle maturation, interrupted the estrous cycle, induced oviductal and uterine hypotrophy. Estrous cyclicity was reestablished 60 days after AAS treatment. However, TC-treated mice still exhibited impaired reproductive capacity, a disturbance potentially related to deficiency in folliculogenesis, sex hormones production, and endometrial receptivity mediate by ER-α, PR, HOXA-10 and LIF down-regulation. Taken together, our findings indicated that in addition to female behavior, reproductive organs microstructure and function are markedly impaired by TC in a dose-dependent manner, whose time-dependent reversibility remains to be clarified.

Schistosoma mansoni co-infection modulates Chagas disease development but does not impair the effect of benznidazole-based chemotherapy.

The adequate management of parasite co-infections represents a challenge that has not yet been overcome, especially considering that the pathological outcomes and responses to treatment are poorly understood. Thus, this study aimed to evaluate the impact of Schistosoma mansoni infection on the efficacy of benznidazole (BZN)-based chemotherapy in Trypanosoma cruzi co-infected mice. BALB/c mice were maintained uninfected or co-infected with S. mansoni and T. cruzi, and were untreated or treated with BZN. Body weight, mortality, parasitemia, cardiac parasitism, circulating cytokines (Th1/Th2/Th17); as well as heart, liver and intestine microstructure were analyzed. The parasitemia peak was five times higher and myocarditis was more severe in co-infected than T. cruzi-infected mice. After reaching peak, parasitemia was effectively controlled in co-infected animals. BZN successfully controlled parasitemia in both co-infected and T. cruzi-infected mice and improved body mass, cardiac parasitism, myocarditis and survival in co-infected mice. Co-infection dampened the typical cytokine response to either parasite, and BZN reduced anti-inflammatory cytokines in co-infected mice. Despite BZN normalizing splenomegaly and liver cellular infiltration, it exacerbated hepatomegaly in co-infected mice. Co-infection or BZN exerted no effect on hepatic granulomas, but increased pulmonary and intestinal granulomas. Marked granulomatous inflammation was identified in the small intestine of all schistosomiasis groups. Taken together, our findings indicate that BZN retains its therapeutic efficacy against T. cruzi infection even in the presence of S. mansoni co-infection, but with organ-specific repercussions, especially in the liver.

Matrix metalloproteinases inhibition reveals the association between inflammation, collagen accumulation and intestinal translocation of Schistosoma mansoni eggs in vivo.

Schistosomiasis mansoni is a parasitic infection that causes enterohepatic morbidity associated with severe granulomatous inflammation triggered by parasite eggs. In this disease, granulomatous inflammation leads to intestinal erosion and environmental excretion of S. mansoni eggs from feces, an essential process for propagating the parasite and infecting host organisms. Metalloproteinases (MMP) are involved in S. mansoni-induced hepatic granulomatous inflammation and fibrosis. However, the relationship between MMP and collagen accumulation with the intestinal excretion of parasite eggs remains unclear. Thus, the present study investigated whether MMP inhibition is capable of modulating granulomatous inflammation, collagen accumulation and mechanical resistance to the point of influencing the dynamics between intestinal retention and excretion of S. mansoni eggs in infected mice. Our findings indicated that doxycycline (a potent MMP inhibitor) aggravates intestinal inflammation and subverts collagen dynamics in schistosomiasis. By attenuating MMP-2 and MMP-9 activity, this drug is capable of enhancing fibrosis and mechanical resistance of the intestinal wall, hindering S. mansoni eggs translocation. Although collagen content was not correlated with MMP activity, intestinal retention and fecal excretion of parasite eggs in untreated mice; these correlations were observed for doxycycline-treated animals. Thus, our study provides evidence that doxycycline is able to attenuate fecal elimination of S. mansoni eggs by inhibiting MMP-2 and MMP-9 activity, events potentially associated with excessive collagen accumulation, which increases intestinal mechanical resistance and hinders eggs translocation through the intestinal wall. Variations in intestinal collagen dynamics are relevant since they may represent changes in the environmental dispersion of S. mansoni eggs, bringing repercussions for schistosomiasis propagation.

Pharmacological potential of new metronidazole/eugenol/dihydroeugenol hybrids against Trypanosoma cruzi in vitro and in vivo.

AIMS: From well-delimited immunomodulatory, redox and antimicrobial properties; metronidazole and eugenol were used as structural platforms to assembly two new molecular hybrids (AD06 and AD07), whose therapeutic relevance was analyzed on T. cruzi infection in vitro and in vivo. METHODS: Non-infected, T. cruzi-infected H9c2 cardiomyocytes, and mice non-treated and treated with vehicle, benznidazole (Bz - reference drug), AD06 and AD07 were investigated. Parasitological, prooxidant, antioxidant, microstructural, immunological, and hepatic function markers were analyzed. RESULTS: Our findings indicated that in addition to having a direct antiparasitic effect on T. cruzi, metronidazole/eugenol hybrids (especially AD07) attenuated cellular parasitism, reactive species biosynthesis and oxidative stress in infected cardiomyocytes in vitro. Although AD06 and AD07 exerted no relevant impact on antioxidant enzymes activity (CAT, SOD, GR and GPx) in host cells, these drugs (especially AD07) attenuated trypanothione reductase activity in T. cruzi, which increased parasite's susceptibility to in vitro pro-oxidant challenge. AD06 and AD07 were well tolerated and do not determine humoral response suppression, mortality (100 % survival) or hepatotoxicity in mice, as indicated by transaminases plasma levels. AD07 also induced relevant in vivo antiparasitic and cardioprotective effects, attenuating parasitemia, cardiac parasite load and myocarditis in T. cruzi-infected mice. Although this cardioprotective response is potentially related to AD07 antiparasitic effect, a direct anti-inflammatory potential of this molecular hybrid cannot be ruled out. CONCLUSION: Taken together, our findings indicated that the new molecular hybrid AD07 stood out as a potentially relevant candidate for the development of new, safe and more effective drug regimens for T. cruzi infection treatment.

4-nitrobenzoylcoumarin potentiates the antiparasitic, anti-inflammatory and cardioprotective effects of benznidazole in a murine model of acute Trypanosoma cruzi infection.

The anti-inflammatory and cardioprotective potential of coumarin metabolites in infectious myocarditis remains overlooked. Thus, the impact of the synthetic 4-nitrobenzoylcoumarin (4NB) alone and combined with benznidazole (Bz) in a murine model of Trypanosoma cruzi-induced acute myocarditis was investigated. Swiss mice infected with T. cruzi were randomized in 8 groups: uninfected, infected untreated or treated with 50 and 100 mg/kg 4NB or Bz alone and combined. Treatments were administered by gavage for 20 days. Cytokines (IL-2, IL-6, IL-10, IL-17, TNFα, and IFN-γ), immunoglobulin reactivity index (total IgG, IgG1, IgG2a and IgG2b), atrial natriuretic peptide (ANP), parasitemia, serum transaminases, heart and liver cellularity were analyzed. T. cruzi infection induced blood parasitism, heart and liver inflammation, upregulated all cytokines, IgG reactivity index, ANP and transaminase levels, determining 43% mortality in untreated mice. Transaminase levels, mean parasitemia, heart inflammation and ANP were reduced in 4NB-treated mice, reaching a 100% survival rate. Total survival (100%) was also obtained in all combinations of Bz and 4NB, which were effective in reducing blood parasitism, transaminases, cytokines and ANP levels, IgG reactivity index, liver and heart interstitial cellularity compared to 50 mg/kg Bz. Our findings indicated that 4NB alone and combined with Bz was well tolerated, showing no evidence of hepatotoxicity. Mainly in combination, these drugs exerted protective effects against T. cruzi-induced acute myocarditis by attenuating blood parasitism, systemic and heart inflammation. Thus, combinations based on 4NB and Bz are potentially relevant to develop new and more effective drug regimens for the treatment of T. cruzi-induced myocarditis.

Chronic rapamycin pretreatment modulates arginase/inducible nitric oxide synthase balance attenuating aging-dependent susceptibility to Trypanosoma cruzi infection and acute myocarditis.

Considering the efficacy of rapamycin in increasing lifespan and healthspan, attenuating the aging-dependent immunological decline, we compared the evolution of Trypanosoma cruzi infection and acute myocarditis in young and elderly mice untreated and chronically treated with this drug. Five groups were investigated: young uninfected and infected, elderly uninfected and infected with Trypanosoma cruzi untreated and treated with rapamycin (4 mg/kg every 3 days) from the 8th to the 96th week of age. Seven days after the last treatment, elderly mice were inoculated with T. cruzi. Young animals were infected at 8-weeks-old. Untreated elderly mice exhibited increase parasitemia, parasite load and myocarditis, which were associated to down-regulation in IL-2, IL-6, IFN-γ, TNF, anti-T. cruzi immunoglobulin G (IgG) total, IgG1 and IgG2a plasma levels, inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) cardiac production, as well as upregulation in Arginase-1 gene expression and arginase activity compared to young animals. These parameters were improved in rapamycin-pretreated elderly mice, which exhibited a better parasitological control, reduced heart inflammation and microstructural damage. These responses were associated with a better balance between Th1 and Th2 effectors similar to that observed in young animals, including an improved activation of Th1 cytokines and the iNOS pathway that positively regulates NO biosynthesis, contradicting the predominant activation of the arginase pathway in untreated elderly animals. Thus, our findings suggest that chronic pretreatment with rapamycin can attenuate immunosenescence in mice, contributing to prolong parasite resistance and attenuate acute myocarditis in elderly host challenged by T. cruzi.

Doxycycline hyclate stimulates inducible nitric oxide synthase and arginase imbalance, potentiating inflammatory and oxidative lung damage in schistosomiasis.

BACKGROUND: We investigated the relationship between inducible nitric oxide synthase (iNOS) and arginase pathways, cytokines, macrophages, oxidative damage and lung granulomatous inflammation in S. mansoni-infected and doxycycline-treated mice. METHODS: Swiss mice were randomized in four groups: (i) uninfected, (ii) infected with S. mansoni, (iii) infected + 200 mg/kg praziquantel (Pzt), (iv) and (v) infected + 5 and 50 mg/kg doxycycline. Pzt (reference drug) was administered in a single dose and doxycycline for 60 days. RESULTS: S. mansoni-infection determined extensive lung inflammation, marked recruitment of M2 macrophages, cytokines (IL-4, IL-5, IFN-γ, TNF-α) upregulation, intense eosinophil peroxidase (EPO) levels, arginase expression and activity, reduced iNOS expression and nitric oxide (NO) production. The higher dose of doxycycline aggravated lung granulomatous inflammation, downregulating IL-4 levels and M2 macrophages recruitment, and upregulating iNOS expression, EPO, NO, IFN-γ, TNF-α, M1 macrophages, protein carbonyl and malondialdehyde tissue levels. The number and size of granulomas in doxycycline-treated animals was higher than untreated and Pzt-treated mice. Exudative/productive granulomas were predominant in untreated and doxycycline-treated animals, while fibrotic/involutive granulomas were more frequent in Pzt-treated mice. The reference treatment with Pzt attenuated all these parameters. CONCLUSION: Our findings indicated that doxycycline aggravated lung granulomatous inflammation in a dose-dependent way. Although Th1 effectors are protective against several intracellular pathogens, effective schistosomicidal responses are dependent of the Th2 phenotype. Thus, doxycycline contributes to the worsening of lung granulomatous inflammation by potentiating eosinophils influx and downregulating Th2 effectors, reinforcing lipid and protein oxidative damage in chronic S. mansoni infection.

Doxycycline Hyclate Modulates Antioxidant Defenses, Matrix Metalloproteinases, and COX-2 Activity Accelerating Skin Wound Healing by Secondary Intention in Rats.

The main objective of this study was to investigate the action of doxycycline hyclate (Dx) in the skin wound healing process in Wistar rats. We investigated the effect of Dx on inflammatory cell recruitment and production of inflammatory mediators via in vitro and in vivo analysis. In addition, we analyzed neovascularization, extracellular matrix deposition, and antioxidant potential of Dx on cutaneous repair in Wistar rats. Male animals (n = 15) were divided into three groups with five animals each (protocol: 72/2017), and three skin wounds (12 mm diameter) were created on the back of the animals. The groups were as follows: C, received distilled water (control); Dx1, doxycycline hyclate (10 mg/kg/day); and Dx2, doxycycline hyclate (30 mg/kg/day). The applications were carried out daily for up to 21 days, and tissues from different wounds were removed every 7 days. Our in vitro analysis demonstrated that Dx led to macrophage proliferation and increased N-acetyl-ß-D-glucosaminidase (NAG) production, besides decreased cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and metalloproteinases (MMP), which indicates that macrophage activation and COX-2 inhibition are possibly regulated by independent mechanisms. In vivo, our findings presented increased cellularity, blood vessels, and the number of mast cells. However, downregulation was observed in the COX-2 and PGE2 expression, which was limited to epidermal cells. Our results also showed that the downregulation of this pathway benefits the oxidative balance by reducing protein carbonyls, malondialdehyde, nitric oxide, and hydrogen peroxide (H2O2). In addition, there was an increase in the antioxidant enzymes (catalase and superoxide dismutase) after Dx exposure, which demonstrates its antioxidant potential. Finally, Dx increased the number of types I collagen and elastic fibers and reduced the levels of MMP, thus accelerating the closure of skin wounds. Our findings indicated that both doses of Dx can modulate the skin repair process, but the best effects were observed after exposure to the highest dose.

Doxycycline aggravates granulomatous inflammation and lung microstructural remodeling induced by Schistosoma mansoni infection.

Although doxycycline exhibits immunomodulatory properties, its effects on pulmonary infection by Schistosoma mansoni remain overlooked. Thus, we investigated the impact of this drug on lung granulomatous inflammation and microstructural remodeling in a murine model of schistosomiasis. Swiss mice were randomized in four groups: (i) uninfected, (ii) infected with S. mansoni and untreated, (iii) infected treated with praziquantel (Pzq; 200 mg/kg), and (iv) infected treated with Dox (50 mg/kg). Pz was administered in a single dose, and Dox for 60 days. S. mansoni induced marked granulomatous lung inflammation, which was associated to cytokines upregulation (IL-2, IL-4, IL-10, IFN-γ, TNF-α, and TGF-ß), neutrophils and macrophages recruitment, alveolar collapse, lung fibrosis, and extensive depletion of elastic fibers. These parameters were attenuated by Pzq and aggravated by Dox. Exudative/productive granulomas were predominant in untreated and Dox-treated animals, while fibrotic granulomas were more frequent in Pzq-treated mice. The number and size of granulomas in Dox-treated animals was higher than untreated and Pzq-treated mice. Dox treatment inhibited the increase in MMP-1 and MMP-2 activity but upregulated myeloperoxidase and N-acetylglucosaminidase activity compared to untreated and Pzq-treated animals. Dox and Pzq exerted no effect on elastin depletion and upregulation of elastase activity. Together, our findings indicated that Dox aggravated granulomatous inflammation, accelerating lung microstructural remodeling by downregulating MMP-1 and MMP-2 activity without impair neutrophils and macrophages recruitment or elastase activity. Thus, Dox potentiates inflammatory damage associated with lung fibrosis, elastin depletion and massive alveolar collapse, profoundly subverting lung structure in S. mansoni-infected mice.

Impact of diminazene aceturate on renin-angiotensin system, infectious myocarditis and skeletal myositis in mice: An in vitro and in vivo study.

Although renin-angiotensin system (RAS) imbalance is manifested in cardiomyopathies with different etiologies, the impact of RAS effectors on Chagas cardiomyopathy and skeletal myositis is poorly understood. Given that diminazene aceturate (DMZ) shares trypanocidal, angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) stimulatory effects, we investigated the impact of DMZ on cardiomyocytes infection in vitro, renin-angiotensin system, Chagas cardiomyopathy and skeletal myositis in vivo. Cardiomyocytes and T. cruzi were used to evaluate DMZ toxicity in vitro. The impact of 20-days DMZ treatment (1 mg/kg) was also investigated in uninfected and T. cruzi-infected mice as follows: control uninfected and untreated, uninfected treated with DMZ, infected untreated and infected treated with DMZ. DMZ had low toxicity on cardiomyocytes, induced dose-dependent antiparasitic activity on T. cruzi trypomastigotes, and reduced parasite load but not infection rates in cardiomyocytes. DMZ increased ACE2 activity and angiotensin-(1-7) plasma levels but exerted no interference on angiotensin-converting enzyme (ACE) activity, ACE, ACE2 and angiotensin II levels in uninfected and infected mice. DMZ treatment also reduced IFN-γ and IL-2 circulating levels but was ineffective in attenuating parasitemia, MCP-1, IL-10, anti-T. cruzi IgG, nitrite/nitrate and malondialdehyde production, myocarditis and skeletal myositis compared to infected untreated animals. As the antiparasitic effect of DMZ in vitro did not manifest in vivo, this drug exhibited limited relevance to the treatment of Chagas disease. Although DMZ is effective in upregulating angiotensin-(1-7) levels, this molecule does not act as a potent modulator of T. cruzi infection, which can establish heart and skeletal muscle parasitism, lipid oxidation and inflammatory damage, even in the presence of high concentrations of this RAS effector.

Thioridazine aggravates skeletal myositis, systemic and liver inflammation in Trypanosoma cruzi-infected and benznidazole-treated mice.

While thioridazine (Tio) inhibits the antioxidant defenses of Trypanosoma cruzi, the gold standard antitrypanosomal drug benznidazole (Bz) has potent anti-inflammatory and pro-oxidant properties. The combination of these drugs has never been tested to determine the effect on T. cruzi infection. Thus, we compared the impact of Tio and Bz, administered alone and in combination, on the development of skeletal myositis and liver inflammation in T. cruzi-infected mice. Swiss mice were randomized into six groups: uninfected untreated, infected untreated, treated with Tio (80 mg/kg) alone, Bz (50 or 100 mg/kg) alone, or a combination of Tio and Bz. Infected animals were inoculated with a virulent T. cruzi strain (Y) and treated by gavage for 20 days. Mice untreated or treated with Tio alone developed the most intense parasitemia, highest parasitic load, elevated IL-10, IL-17, IFN-γ, and TNF-α plasma levels, increased N-acetylglucosaminidase and myeloperoxidase activity in the liver and skeletal muscle, as well as severe myositis and liver inflammation (P < 0.05). All parameters were markedly attenuated in animals receiving Bz alone (P < 0.05). However, the co-administration of Tio impaired the response to Bz chemotherapy, causing a decrease in parasitological control (parasitemia and parasite load), skeletal muscle and liver inflammation, and increased microstructural damage, when compared to the group receiving Bz alone (P < 0.05). Altogether, our findings indicated that Tio aggravates systemic inflammation, skeletal myositis and hepatic inflammatory damage in T. cruzi-infected mice. By antagonizing the antiparasitic potential of Bz, Tio limits the anti-inflammatory, myoprotectant and hepatoprotective effects of the reference chemotherapy, aggravating the pathological remodeling of both organs. As the interaction of T. cruzi infection, Bz and Tio is potentially toxic to the liver, inducing inflammation and microvesicular steatosis; this drug combination represents a worrying pharmacological risk factor in Chagas disease.

Could phenothiazine-benznidazole combined chemotherapy be effective in controlling heart parasitism and acute infectious myocarditis?

Phenothiazines inhibit major antioxidant defense mechanisms in trypanosomatids and exhibit potent cytotoxic effects in vitro. However, the relevance of these drugs in the treatment of Trypanosoma cruzi-induced acute myocarditis is poorly explored, especially in combination with reference trypanocidal drugs. Thus, we compared the antiparasitic and cardioprotective potential of thioridazine (TDZ) and benznidazole (Bz) administered in monotherapy and combined in a murine model of T. cruzi-induced acute myocarditis. Female mice were randomized into six groups: (i) uninfected untreated, (ii) infected untreated, or infected treated with (iii) Bz (100 mg/kg), (iv) TDZ (80 mg/kg), (v) Bz (100 mg/kg) + TDZ (80 mg/kg), or (vi) Bz (50 mg/kg) + TDZ (80 mg/kg). Infected animals were inoculated with 2000 T. cruzi trypomastigotes and treated by gavage for 20 days. Animals that received TDZ alone presented the highest levels of parasitemia, parasitic load and anti-T. cruzi immunoglobulin G titers; cardiac upregulation of N-acetyl-ß-D-glucosaminidase activity, nitric oxide, malondialdehyde and cytokines (IFN-γ, TNF-α, IL-10 and IL-17); as well as microstructural damage compared to the other groups (p < 0.05). These parameters were reduced in groups receiving Bz monotherapy compared to the other groups (p < 0.05). The combination of TDZ and Bz attenuated the response to treatment, worsening parasitological control, oxidative heart damage and myocarditis compared to the group treated with Bz alone (p < 0.05). Our results indicate that when administered alone, TDZ potentiated the pathological outcomes in animals infected with T. cruzi. Moreover, TDZ attenuated the antiparasitic effect of Bz when administered together, impairing parasitological control, potentiating inflammation, molecular oxidation and pathological microstructural remodeling of the heart. Thus, our findings indicate that TDZ acts as a pharmacological risk factor and Bz-based monotherapy remains a better cardioprotective drug against Trypanosoma cruzi-induced acute myocarditis.

Sesquiterpene lactone potentiates the immunomodulatory, antiparasitic and cardioprotective effects on anti-Trypanosoma cruzi specific chemotherapy.

We investigated the immunomodulatory, antiparasitic and cardioprotective effects of a sesquiterpene lactone (SL) administered alone or combined with benznidazole (Bz), in a murine model of Chagas' disease by in vitro and in vivo assays. Antiparasitic and cytotoxic potential of tagitinin C (SL) and Bz were tested in vitro against T. cruzi epimastigotes and cardiomyocytes. Swiss mice challenged with T. cruzi were also treated for 20 days with tagitinin C (10 mg/kg) alone and combined with Bz (100 mg/kg). Tagitinin C exhibited a higher antiparasitic (IC50: 1.15 µM) and cytotoxic (CC50 at 6.54 µM) potential than Bz (IC50: 35.81 µM and CC50: 713.5 µM, respectively). When combined, these drugs presented an addictive interaction, determining complete suppression of parasitemia and parasitological cure in all infected mice (100%) compared to those receiving Bz alone (70%). Anti-T. cruzi immunoglobulin G, and pro-inflammatory cytokines IFN-γ and TNF-α levels were reduced in animals treated with tagitinin C combined with Bz, while IL-10 production was unaffected. Heart inflammation was undetectable in 90% of the animals receiving this combination, while only 50% of the animals receiving Bz alone showed no evidence of myocarditis. Together, our findings indicated that the combination of tagitinin C and Bz exerts potent antiparasitic, immunomodulatory and cardioprotective effects. Due to the remarkable suppression of parasitemia and high parasitological cure, this combination was superior to Bz monotherapy, indicating a high potential for the treatment of Chagas's disease.