Inhibition of Tityus serrulatus venom hyaluronidase affects venom biodistribution

Abstract

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.

Fig 1. In vitro neutralization assay using rabbit anti-hyaluronidase serum.

Hyaluronidase activity (%) was measured using a turbidimetric assay. Commercial hyaluronidase from bovine testis was used as a positive control, and ultrapure water was used as a negative control. Enzymatic activities of TsV (2 μg) and native hyaluronidase from TsV (0.5 μg) were tested. For the in vitro neutralization assay, TsV (2 μg) was incubated with pre-immune serum (10 μl) or anti-hyaluronidase serum (Anti-Hyal, 10 μl) for 1 h at 37°C before testing. Anti-hyaluronidase serum neutralized the hyaluronidase activity in TsV. All values are expressed as the mean ± S.E.M. of duplicates from three independent experiments.

Fig 2. In vitro stability of ^99mTc-TsV.

Stability of the complex ^99mTc-TsV over time in the presence of saline 0.9% (w/v) at room temperature and in the presence of plasma at 37°C. All values are presented as the mean ± S.E.M. of duplicates from three independent experiments.

Fig 3. Blood clearance of 99mTc-TsV. 3.7 MBq of 99mTc-TsV diluted in PBS (99mTc-TsV + PBS) or neutralized with anti-hyaluronidase serum (99mTc-TsV + Anti-Hyal serum) was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group).

Radioactivity levels were measured in blood samples at 1, 5, 10, 15, 20, 30, 45, 60, 90, 120, 240, and 1440 min post-injection. Data are represented as the mean percentage of the injected dose of 99mTc-TsV per gram of blood (% ID/g) ± S.E.M. of the mean. Values represent duplicates from two independent experiments. Statistical analyses were performed using a linear mixed model. Serum administration (p < 0.0001), time (p < 0.0001), and their interaction (p < 0.0001) had a statistically relevant effect on the mean 99mTc-TsV blood clearance.

Fig 4. ^99mTc-TsV spreading in mice over time.

Representative scintigraphic images of mice injected with 18 MBq ^99mTc-TsV diluted in PBS (A) or neutralized with anti-hyaluronidase serum (B). Samples were intravenously injected in Swiss mice (6–8 weeks old, 24–28 g; n = 3 per group). Radioactivity levels were measured 30, 60, and 120 min post-injection. Images show a quick and growing spread of ^99mTc-TsV diluted in PBS over time (A). On the other hand, TsV neutralized with anti-hyaluronidase serum remains at the injection site (right tight muscle) (B). Images are pseudocolored according to the color scale.

Fig 5. Biodistribution of ^99mTc-TsV.

^99mTc-TsV (3.7 MBq) diluted in PBS (^99mTc-TsV + PBS) or neutralized with anti-hyaluronidase serum (^99mTc-TsV + Anti-Hyal serum) was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group). Radioactivity levels were measured in the heart, liver, lungs, spleen, thyroid and kidneys at 30, 60, 240 and 1440 min post-injection. The results are expressed as the percentage of injected dose/g of tissue (%ID/g). All values are presented as the mean ± S.E.M. of two independent experiments. Statistical analysis was performed using two-way ANOVA (factors: serum administration and time). Anti-hyaluronidase serum significantly affected the mean distribution of TsV to the liver (p < 0.0001), spleen (p = 0.0115), and kidneys (p = 0.0009), while time was a significant factor for TsV distribution to the heart (p < 0.0001), liver (p < 0.0001), lungs (p = 0.0095), spleen (p = 0.0008), and kidneys (p < 0.0001). A significant interaction between serum administration and time was observed in the heart (p = 0.00003), liver (p < 0.0001), spleen (p = 0.0337), and thyroid (p < 0.0001).

Fig 6. Hyaluronidase neutralization as a first-aid treatment for scorpion sting.

3.7 MBq of ^99mTc-TsV diluted in PBS was intramuscularly injected in Swiss mice (6–8 weeks old, 24–28 g; n = 6 per group). Subsequently, anti-hyaluronidase serum was injected in the same site of ^99mTc-TsV injection at different times (Anti-Hyal serum; 0, 10, and 30 min post-injection; arrows indicate the injection times). Radioactivity levels in the bloodstream were measured at 1, 5, 10, 15, 20, 30, 45, 60, 90, 120, and 240 min post-injection of ^99mTc-TsV. All data are expressed as the mean percentage of the injected dose of ^99mTc-TsV per gram of blood (% ID/g) ± S.E.M. of the mean. Values are representative of duplicates from two independent experiments. Statistical analysis was performed using a linear mixed model. Time (p < 0.0001) and time x Anti-Hyal serum administration interaction (p < 0.0001) had a statistically relevant effect on the mean ^99mTc-TsV blood clearance.