Hyaluronidase: the spreading factor of Tityus serrulatus venom

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. 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 minutes, 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 minutes 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 minutes 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 minutes 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 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. Author summary Hyaluronidases are known as the venom components responsible for disseminating toxins from the injection site to the victim’s organism. Therefore, understanding how the venom distribution occurs and the role of hyaluronidases in this process is crucial in the field of toxinology. In this study, we inhibited Tityus serrulatus venom (TsV) hyaluronidase’s action using specific anti-Ts-hyaluronidase antibodies. Labeling TsV with a radioactive compound enabled monitoring of its biodistribution in mice. Our results show that, upon hyaluronidase inhibition, TsV remains at the injection site for longer, and only a reduced amount of the venom reaches the bloodstream. Consequently, the venom arrives later at target organs like the heart, liver, lungs, spleen, and thyroid. Considering the possible application of hyaluronidase inhibition as a therapeutic resource in envenoming first-aid treatment, we performed the administration of hyaluronidase neutralizing antibodies at different times after TsV injection. We observed that TsV remains in the bloodstream and its arrival at tissues is delayed by 120 or 240 minutes after TsV injection, depending on anti-hyaluronidase administration times. Our data show that hyaluronidase plays a crucial role in TsV spreading from the injection site to the bloodstream and from the bloodstream to the organs, thus suggesting that its inhibition can help to improve envenoming’s treatment.