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. 2022 May 22;19(10):6294.
doi: 10.3390/ijerph19106294.

Acute, Sublethal, and Developmental Toxicity of Kratom (Mitragyna speciosa Korth.) Leaf Preparations on Caenorhabditis elegans as an Invertebrate Model for Human Exposure

Samantha Hughes  1 , David van de Klashorst  2 , Charles A Veltri  3 , Oliver Grundmann  3   4
Affiliations

Acute, Sublethal, and Developmental Toxicity of Kratom (Mitragyna speciosa Korth.) Leaf Preparations on Caenorhabditis elegans as an Invertebrate Model for Human Exposure

Samantha Hughes et al. Int J Environ Res Public Health. .

Abstract

Kratom (Mitragyna speciosa Korth.) is a tree native to Southeast Asia with stimulant and opioid-like effects which has seen increased use in Europe and North America in recent years. Its safety and pharmacological effects remain under investigation, especially in regard to developmental and generational toxicity. In the current study, we investigated commercial kratom preparations using the nematode Caenorhabditis elegans as a translational model for toxicity and pharmacological effects. The pure alkaloids mitragynine and 7-hydroxymitragynine as well as aqueous, ethanolic, and methanolic extracts of three commercial kratom products were evaluated using a battery of developmental, genotoxic, and opioid-related experiments. As determined previously, the mitragynine and 7-hydroxymitragynine content in kratom samples was higher in the alcoholic extracts than the aqueous extracts. Above the human consumption range equivalent of 15-70 µg/mL, kratom dose-dependently reduced brood size and health of parent worms and their progeny. 7-hydroxymitragynine, but not mitragynine, presented with toxic and developmental effects at very high concentrations, while the positive control, morphine, displayed toxic effects at 0.5 mM. Kratom and its alkaloids did not affect pumping rate or interpump interval in the same way as morphine, suggesting that kratom is unlikely to act primarily via the opioid-signalling pathway. Only at very high doses did kratom cause developmental and genotoxic effects in nematodes, indicating its relative safety.

Keywords: body bending; opioid; pharyngeal pumping; reproduction; toxicity.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
LC-QTOF chromatograms: All panels represent the base peak chromatogram. (A) 7-hydroxymitragynine standard, retention time 2.45 min. (B) The 25 mg/mL Red Maeng Da kratom methanolic extract. (C) The 25 mg/mL Red Maeng Da kratom aqueous extract. (D) Mitragynine standard, retention time 5.31 min.
Figure 2
Figure 2
Acute toxicity of kratom towards the parental worms: The mobility of the parent was observed after 48 h of incubation with the kratom extracts. The worms were classified as ‘healthy’ (green bars), where the worm was freely moving with normal head-to-tail body bends or ‘slightly abnormal’ (yellow bars) where the worm had a defective thrashing motion. Any parental worms that were ‘dead/not moving’ (red bars) were also noted. The percentage of wells with the worms in each category was calculated and is shown as a stacked bar chart. White Borneo, Red Maeng Da, or Bali varieties extracted in water, methanol, or ethanol were added at 15, 25, 45, 70, 90, 120, 150, 200, 250, 300, 350, and 400 µg/mL.
Figure 3
Figure 3
Brood size of C. elegans after 48 h of incubation with kratom: Worms were placed in liquid culture at the L4 stage and incubated at 20 °C for 48 h with shaking at 150 rpm. Kratom was added at 15, 25, 45, 70, 90, 120, 150, 200, 250, 300, 350, and 400 µg/mL with (A) White Borneo, (B) Red Maeng Da, or (C) Bali varieties extracted in ethanol (squares, dashed line), methanol (circles, solid line), or water (triangles, dotted line). The brood size was assessed by binning the data into 4 groups: no viable progeny; up to 10 viable progeny; 11–75 viable progeny; and >75 viable progeny (i.e., wild type). An average of the bins across the replicates was found and plotted with the standard error of the mean (s.e.m.) with the lines indicting the non-linear fit, with a hillslope of −1 using GraphPad Prism. Detailed information can be found in Supplemental Figures S1 and S2.
Figure 4
Figure 4
The effect of mitragynine, 7-hydroxymitragynine, and morphine on C. elegans: Both mitragynine and 7-hydroxymitragynine were tested at 5, 0.5, and 0.1 µg/mL in the assay, with the methanol control of 5%, 0.5%, and 0.1%, respectively. In this case, methanol was the solvent control as mitragynine and 7-hydroxymitragynine are not able to be fully dissolved in DMSO, and C. elegans can tolerate methanol up to 5% [53]. Morphine was only tested at 0.5 mM, as suggested in the literature [40]. (A) The brood size was assessed by binning the data into four groups: no viable progeny, up to 10 viable progeny, 11–75 viable progeny, or more than 75 viable progeny. An average of the bins across the replicates was found and plotted with the standard error of the mean and a non-linear regression line using GraphPad Prism. Mitragynine (circles, solid line) and the methanol control (triangles, dotted line) had no effect on the brood size of the worms. In contrast, 7-hydroxymitrgynine (squares, dashed line) resulted in a striking decrease in brood size at the highest concentration tested, 5 µg/mL. (B) Morphine did result in a decrease in brood, with around half of the replicates displaying a reduction in brood size. The brood size was assessed by binning the data into one of four classes: no viable progeny, red bars; up to 10 viable progeny (dark-grey bars); 11–75 viable progeny (light-grey bars); and more than 75 viable progeny (green bars). The percentage of wells in each category was calculated and plotted. (C) After 48 h of incubation with morphine, mitragynine, 7-hydroxymitrgynine, or methanol, the development of the progeny was assessed. The movement of the progeny was assessed in each well and classified as ‘healthy’ (green bars), ‘slightly abnormal (yellow bars), or ‘dead/not moving’ (red bars). The percentage of wells with offspring in each category at each concentration is shown. (D) The acute toxicity to the parent nematode was assessed after 48 h of incubation with morphine, mitragynine, 7-hydroxymitragynine, or methanol. The movement of the progeny was assessed in each well and classified as ‘healthy’ (green bars), ‘slightly abnormal’ (orange bars), or ‘dead/not moving’ (red bars). The percentage of wells with offspring in each category at each concentration is shown.
Figure 5
Figure 5
Thrashing was reduced in worms exposed to morphine and only the White Borneo variety of kratom: The thrashing of worms was normalised to control conditions (black bar), which was set to 100%. The vehicle control (0.6% DMSO, grey bar) showed no change in thrash rate, while 0.5 mM morphine (white bar) significantly reduced the body bends per minute in nematodes (p < 0.05, indicated by *, ** shows p < 0.01 and *** indicates p < 0.001). Neither Red Maeng Da kratom at 300 µg/mL (green bars) nor the Bali variety (purple bars) had a striking effect on body bends in worms with any of the extracts. There was a difference in the body bends observed when worms were exposed to the White Borneo kratom (blue bars). The water extract resulted in a small but significant decrease in body bends (p = 0.02, shown by *) with exposure to methanolic extracted White Borneo, resulting in a significant reduction in body bending (p = 0.002, shown by **). See Supplemental Figure S3 for individual counts of thrashing.
Figure 6
Figure 6
Morphine, but not kratom, increased the pumping rate in starved animals: Violin plot to show the pumps per minute for worms exposed to White Borneo kratom at 300 µg/mL, 0.5 mM morphine, or the controls. The violin plots also show the median value as a dotted line and statistics as shown from the one-way ANOVA test. (A) Well-fed worms. There are no statistical differences in these conditions. (B) Worms which were starved for 1 h. p-values are shown as a result of one-way ANOVA analysis where * p < 0.05, ** p < 0.005, and *** p < 0.0005.
Figure 7
Figure 7
Representative electropharyngeogram traces of C. elegans exposed to White Borneo kratom with 1 h of starvation: Worms (L4 stage) were added to NGM supplemented with methanol, ethanol, or water extracts of White Borneo kratom (300 µg/mL) or 0.5 mM morphine and incubated at 20 °C. After 48 h, worms were transferred to kratom/morphine-supplemented NGM without food for 1 h before being subjected to analysis in the In Vivo Biosystem ScreenChip ™ system. (A) Representative trace of a control nematode for 50 s of a 2 min recording. The area of the red box, approximately 2 s, is expanded and shown in B. (B) Expanded traces of approximately 2 s showing the individual pumps. (i) Control nematodes; (ii) nematodes exposed to 0.5 mM morphine had more pumps which were smaller and shorter; (iii) Worms exposed to 300 µg/mL methanol extract of White Borneo kratom had pumps which were similar to the control; (iv) ethanol extract of White Borneo kratom at 300 µg/mL, and (v) White Borneo extracted in water had similar pumping profiles as the control animals. Detailed information on the EPGs is displayed in Table 4.
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