Guarana (Paullinia cupana Mart.) attenuates methylmercury-induced toxicity in Caenorhabditis elegans

Abstract

The influence of routine guarana (Paullinia cupana) consumption on apparent tolerance to mercury intoxication has been proposed. The present study investigated this hypothesis in Caenorhabditis elegans, a suitable experimental model for studies in toxicology. Wild type (WT) and skn-1 (ok2315) worm strains were pretreated with guarana ethanolic extract (GEE) from larvae 1 (L1) to L4 stage and then exposed for 6 hours to methylmercury (MeHg). The analyses included evaluation of GEE's effects on lethality, developmental delay, feeding, locomotion, gene expression (sod-3, gst-4, sir-2.1, hsf-1, snn-1, mtl-1, mtl-2, aat-1, aat-2 and aat-3) and antioxidant activity. GEE pre-treatment had no aberrant effects on WT worms exposed to MeHg, and protected skn-1 (ok2315) worms, which are more susceptible to environmental stresses. Protective effects of GEE might be dependent on modulation of genes other than those directly involved in antioxidant activity. GEE increased the expression of genes involved in metal transport (aat-2), metal detoxification (mtl-1 and mtl-2) and antioxidant responses (sir-2.1 and sod-3). Thus, routine consumption of guarana might be beneficial in protecting against MeHg-induced toxicity.

Keywords: mercury; natural products; neurological disorders; xanthines.

Fig. 1. Dose–response curves of C. elegans lethality upon 6-hour MeHg (0–150 μM) exposure and a 24-hour recovery period ± pre-treatment with guarana ethanolic extract (GEE). The L4-larval stage worms untreated and pretreated with GEE from L1 were exposed to methylmercury chloride (CH₃HgCl, MeHgCl) for 6 hours. Following washing, worms were transferred to NGM plates seeded with E. coli OP50, allowed to recover for 24 hours and then scored as dead or alive using a stereomicroscope. GEE had no effect against MeHg-induced lethality on wild type (WT) worms (LD50 ≈ 66 μM) (A) but significantly increased survival in skn-1(ok2315) worms (LD50 = 38, 45, 51 and 59 μM to untreated and GEE pretreated worms at 100, 500 and 1000 μg ml^–1, respectively) (B). Data were compared with the extra sum-of-squares F test method, p < 0.05, and are expressed as means ± SEM, n = 4, approximately 100 worms analyzed per group in each assay.

Fig. 2. Developmental progress of wild type (A) and skn-1 (ok2315) (B) untreated and GEE-pretreated worms post 6-hour MeHg (0–100 μM) exposure and 24-hour recovery. The surviving worms were analyzed using a microscope and scored through the larval stages observing vulva development and the presence of eggs in the uterus. WT and skn-1 (ok2315) control worms reached the egg laying adult-stage, while MeHg exposure induced a dose-dependent delay in worm development starting at 25 μM for both strains, with an accentuated effect in mutant worms. Pretreatment with GEE significantly decreased the percentage of worms delayed only in the mutant strain at 25 (C) and 50 μM of MeHg (D). (#) Represents a significant difference between control and metal-exposed worms by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05) and (*) represents significant difference from the untreated group and (^a–c) represents significant difference between GEE doses by two-way ANOVA followed by Bonferroni's Multiple Comparison Test (p < 0.05). Data are expressed as means ± SEM, n = 4, at least 30 worms analyzed per group in each assay.

Fig. 3. Pharyngeal pumping rate in WT and skn-1 (ok2315) untreated and GEE-pretreated worms at 0 hours (A and B) and 24 hours (C and D) post 6-hour MeHg exposure. The number of pharyngeal contractions per minute was observed in worms in E. coli OP50-seeded NGM plates with a microscope. MeHg induced a dose-dependent decrease in pharyngeal pumping rates immediately post 6-hour exposure and GEE exerted protective effects only in skn-1 (ok2315) worms at 25 μM MeHg. Post 24 hours, the worms showed a recovery in the pharyngeal pumping rate to control levels, with the exception of the 50 μM MeHg group. (#) Represents a significant difference between control and metal exposed worms by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05) and (*) represents a significant difference between untreated and GEE-pretreated worms by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05). Data are expressed as means ± SEM, n = 4, 10 worms analyzed per group in each assay.

Fig. 4. Number of thrashes per minute in WT and skn-1(ok2315) untreated and GEE-pretreated worms 0 hours (A and B) and 24 hours (C and D) post 6-hour MeHg exposure. A thrash was defined as a change in the direction of bending at the middle of the body and was assessed in worms individually placed in a drop of M9 with a stereomicroscope. MeHg induced a dose-dependent decrease in the number of thrashes immediately post 6-hour exposure, and post 24 hours, worms partially recovered movements. (#) Represents a significant difference between control and metal exposed worms by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05) and (*) represents a significant difference between untreated and GEE-pre treated worms by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05). Data are expressed as means ± SEM, n = 4.

Fig. 5. ROS-associated fluorescence levels in WT and skn-1 (ok2315) worms untreated and pretreated with GEE 1000 μg ml^–1 after MeHg exposure and 24-hour recovery. Aliquots of worm's lysates were incubated for 1 hour with 20 μM DCFDA (final concentration) at 20 °C and fluorescence was measured in duplicate in a microplate reader at 485 nm excitation and 520 nm emission wavelengths at room temperature. Data were normalized to protein content. Fluorescence levels were significantly increased in MeHg non-exposed groups in comparison to control and MeHg at 25 and 50 μM significantly decreased fluorescence levels in comparison to MeHg-non-exposed worms of the same group. No significant differences were detected between untreated and GEE-pretreated worms and between WT and skn-1 (ok2315) worms exposed to MeHg. (#) Represents a significant difference between control and the other groups non-exposed to MeHg by two-way ANOVA followed by Bonferroni's multiple comparison test (p < 0.05) and (*) represents a significant difference between MeHg-exposed and non-exposed worms of the same group by one-way ANOVA followed by the Bonferroni post hoc test (p < 0.05). Data are expressed as means of arbitrary fluorescence units (AFU) per μg of protein ± SEM, n = 4.