Inhibition of Tityus serrulatus venom hyaluronidase affects venom biodistribution.

BACKGROUND: The hyaluronidase enzyme is generally known as a spreading factor in animal venoms. Although its activity has been demonstrated in several organisms, a deeper knowledge about hyaluronidase and the venom spreading process from the bite/sting site until its elimination from the victim's body is still in need. Herein, we further pursued the goal of demonstrating the effects of inhibition of T. serrulatus venom (TsV) hyaluronidase on venom biodistribution. METHODS AND PRINCIPAL FINDINGS: We used technetium-99m radiolabeled Tityus serrulatus venom (99mTc-TsV) to evaluate the venom distribution kinetics in mice. To understand the hyaluronidase's role in the venom's biodistribution, 99mTc-TsV was immunoneutralized with specific anti-T.serrulatus hyaluronidase serum. Venom biodistribution was monitored by scintigraphic images of treated animals and by measuring radioactivity levels in tissues as heart, liver, lungs, spleen, thyroid, and kidneys. In general, results revealed that hyaluronidase inhibition delays venom components distribution, when compared to the non-neutralized 99mTc-TsV control group. Scintigraphic images showed that the majority of the immunoneutralized venom is retained at the injection site, whereas non-treated venom is quickly biodistributed throughout the animal's body. At the first 30 min, concentration peaks are observed in the heart, liver, lungs, spleen, and thyroid, which gradually decreases over time. On the other hand, immunoneutralized 99mTc-TsV takes 240 min to reach high concentrations in the organs. A higher concentration of immunoneutralized 99mTc-TsV was observed in the kidneys in comparison with the non-treated venom. Further, in situ neutralization of 99mTc-TsV by anti-T.serrulatus hyaluronidase serum at zero, ten, and 30 min post venom injection showed that late inhibition of hyaluronidase can still affect venom biodistribution. In this assay, immunoneutralized 99mTc-TsV was accumulated in the bloodstream until 120 or 240 min after TsV injection, depending on anti-hyaluronidase administration time. Altogether, our data show that immunoneutralization of hyaluronidase prevents venom spreading from the injection site. CONCLUSIONS: By comparing TsV biodistribution in the absence or presence of anti-hyaluronidase serum, the results obtained in the present work show that hyaluronidase has a key role not only in the venom spreading from the inoculation point to the bloodstream, but also in venom biodistribution from the bloodstream to target organs. Our findings demonstrate that hyaluronidase is indeed an important spreading factor of TsV and its inhibition can be used as a novel first-aid strategy in envenoming.

Cardiovascular-Active Venom Toxins: An Overview.

Animal venoms are a mixture of bioactive compounds produced as weapons and used primarily to immobilize and kill preys. As a result of the high potency and specificity for various physiological targets, many toxins from animal venoms have emerged as possible drugs for the medication of diverse disorders, including cardiovascular diseases. Captopril, which inhibits the angiotensin-converting enzyme (ACE), was the first successful venom-based drug and a notable example of rational drug design. Since captopril was developed, many studies have discovered novel bradykinin-potentiating peptides (BPPs) with actions on the cardiovascular system. Natriuretic peptides (NPs) have also been found in animal venoms and used as template to design new drugs with applications in cardiovascular diseases. Among the anti-arrhythmic peptides, GsMTx-4 was discovered to be a toxin that selectively inhibits the stretch-activated cation channels (SACs), which are involved in atrial fibrillation. The present review describes the main components isolated from animal venoms that act on the cardiovascular system and presents a brief summary of venomous animals and their venom apparatuses.

Molecular, immunological, and biological characterization of Tityus serrulatus venom hyaluronidase: new insights into its role in envenomation.

BACKGROUND: Scorpionism is a public health problem in Brazil, and Tityus serrulatus (Ts) is primarily responsible for severe accidents. The main toxic components of Ts venom are low-molecular-weight neurotoxins; however, the venom also contains poorly characterized high-molecular-weight enzymes. Hyaluronidase is one such enzyme that has been poorly characterized. METHODS AND PRINCIPAL FINDINGS: We examined clones from a cDNA library of the Ts venom gland and described two novel isoforms of hyaluronidase, TsHyal-1 and TsHyal-2. The isoforms are 83% identical, and alignment of their predicted amino acid sequences with other hyaluronidases showed conserved residues between evolutionarily distant organisms. We performed gel filtration followed by reversed-phase chromatography to purify native hyaluronidase from Ts venom. Purified native Ts hyaluronidase was used to produce anti-hyaluronidase serum in rabbits. As little as 0.94 µl of anti-hyaluronidase serum neutralized 1 LD50 (13.2 µg) of Ts venom hyaluronidase activity in vitro. In vivo neutralization assays showed that 121.6 µl of anti-hyaluronidase serum inhibited mouse death 100%, whereas 60.8 µl and 15.2 µl of serum delayed mouse death. Inhibition of death was also achieved by using the hyaluronidase pharmacological inhibitor aristolochic acid. Addition of native Ts hyaluronidase (0.418 µg) to pre-neutralized Ts venom (13.2 µg venom+0.94 µl anti-hyaluronidase serum) reversed mouse survival. We used the SPOT method to map TsHyal-1 and TsHyal-2 epitopes. More peptides were recognized by anti-hyaluronidase serum in TsHyal-1 than in TsHyal-2. Epitopes common to both isoforms included active site residues. CONCLUSIONS: Hyaluronidase inhibition and immunoneutralization reduced the toxic effects of Ts venom. Our results have implications in scorpionism therapy and challenge the notion that only neurotoxins are important to the envenoming process.