Irinotecan and Δ⁸-Tetrahydrocannabinol Interactions in Rat Liver: A Preliminary Evaluation Using Biochemical and Genotoxicity Markers

Abstract

There is growing interest regarding the use of herbal preparations based on Cannabis sativa for medicinal purposes, despite the poorly understood interactions of their main constituent Δ⁸-tetrahydrocannabinol (THC) with conventional drugs, especially cytostatics. The objective of this pilot study was to prove whether the concomitant intake of THC impaired liver function in male Wistar rats treated with the anticancer drug irinotecan (IRI), and evaluate the toxic effects associated with this exposure. IRI was administered once intraperitoneally (at 100 mg/kg of the body weight (b.w.)), while THC was administered per os repeatedly for 1, 3, and 7 days (at 7 mg/kg b.w.). Functional liver impairments were studied using biochemical markers of liver function (aspartate aminotransferase-AST, alanine aminotransferase-ALP, alkaline phosphatase-AP, and bilirubin) in rats given a combined treatment, single IRI, single THC, and control groups. Using common oxidative stress biomarkers, along with measurement of primary DNA damage in hepatocytes, the degree of impairments caused at the cellular level was also evaluated. THC caused a time-dependent enhancement of acute toxicity in IRI-treated rats, which was confirmed by body and liver weight reduction. Although single THC affected ALP and AP levels more than single IRI, the levels of liver function markers measured after the administration of a combined treatment mostly did not significantly differ from control. Combined exposure led to increased oxidative stress responses in 3- and 7-day treatments, compared to single IRI. Single IRI caused the highest DNA damage at all timepoints. Continuous 7-day oral exposure to single THC caused an increased mean value of comet tail length compared to its shorter treatments. Concomitant intake of THC slightly affected the levels of IRI genotoxicity at all timepoints, but not in a consistent manner. Further studies are needed to prove our preliminary observations, clarify the underlying mechanisms behind IRI and THC interactions, and unambiguously confirm or reject the assumptions made herein.

Keywords: cannabinoid-based preparations; functional liver impairments; genotoxicity; hepatocytes; liver to body weight ratio; oxidative stress.

Figure 1

Structural formulas of irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), and experimental schedule. Legend: i.p., intraperitoneal; p.o., per os; D, day; −, without treatment; ↑, termination of the experiment (sampling).

Figure 2

Changes in body weight gains in male Wistar rats after the 1-, 3-, and 7-day treatments with IRI, THC, their combination (IRI + THC), and in the respective controls. Values are expressed as mean ± SD (N = 5). Significantly different (p < 0.05) values were c: vs. control; t: vs. THC.

Figure 3

Liver weight changes in male Wistar rats after the 1-, 3-, and 7-day treatments with IRI, THC, their combination (IRI + THC), and in the respective controls. Values are expressed as mean ± SD (n = 5). Significantly different (p < 0.05; ANOVA, post hoc Tukey HSD test) values were c: vs. control; t: vs. THC; 1: vs. 1-day treatment.

Figure 4

Liver to body weight ratio in male Wistar rats after the 1-, 3-, and 7-day treatments with IRI, THC, their combination (IRI + THC), and in the respective controls. Values are expressed as mean ± SD (n = 5). Significantly different (p < 0.05; ANOVA, post hoc Tukey HSD test) values were c: vs. control; 3: vs. 3-day treatment.

Figure 5

Effects of 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), and their combination (IRI + THC) on aspartate aminotransferase (AST) activity in the serum of male Wistar rats. Values are expressed as mean ± SD (n = 5). The significantly different (p < 0.05) value was c: vs. control rats.

Figure 6

Effects of 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), and their combination (IRI + THC) on alanine aminotransferase (ALT) activity in the serum of male Wistar rats. Values are expressed as mean ± SD (n = 5). The significantly different (p < 0.05) value was i: vs. IRI-treated rats.

Figure 7

Effects of 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC) on alkaline phosphatase (ALP) activity in the serum of male Wistar rats. Values are expressed as mean ± SD (n = 5). The significantly different (p < 0.05) value was c: vs. control rats.

Figure 8

Effects of 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), and their combination (IRI + THC) on total protein in the serum of male Wistar rats. Values are expressed as mean ± SD (n = 5). The significantly different (p < 0.05) value was c: vs. control rats.

Figure 9

Serum bilirubin levels measured in male Wistar rats after the 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC), and in the respective controls. Values are expressed as mean ± SD (n = 5). The significantly different (p < 0.05) values were c: vs. control; i: vs. IRI-treated rats; t: vs. THC-treated rats; &: vs. rats treated with IRI and THC.

Figure 10

Changes in the thiobarbituric reactive substances (TBARS) concentration in the liver of rats after the 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC), and in the respective controls. The results are shown as median and interquartile range. The significantly increased values (p < 0.05) were (a) compared to rats treated with IRI.

Figure 11

Changes in superoxide dismutase (SOD) activity in the liver of rats after the 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC), and in the respective controls. The results are shown as median and interquartile range. The significantly increased values (p < 0.05) were: (*) compared to control, (b) compared to rats treated with THC.

Figure 12

Changes in catalase (CAT) activity in the liver of rats after the 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC), and in the respective controls. The results are shown as median and interquartile range. The significantly increased values (p < 0.05) were (*) compared to control, (a) compared to rats treated with IRI, (b) compared to rats treated with THC.

Figure 13

The levels of primary DNA damage measured in hepatocytes of male Wistar rats using the alkaline comet assay after 1-, 3-, and 7-day treatments with irinotecan (IRI), Δ⁹-tetrahydrocannabinol (THC), their combination (IRI + THC), and in the respective controls. Legend: IRI was administered once, via an intraperitoneal injection at a dose of 100 mg/kg b.w.; THC was dissolved in sesame oil and administered daily per os at a dose of 7 mg/kg b.w.; control rats were given the same daily volume of sesame oil as the THC group. Data are reported as mean/median values, and ranges (Min.—minimum value; Max.—maximum value) of one thousand independent comet measurements per each experimental group (200 comets/rat were scored on duplicate slides). Significantly different (p < 0.05, ANOVA with post hoc Tukey HSD test) compared to C—control; T—THC; &—combination of IRI and THC; I—1-day treatment; II—3-day treatment; III—7-day treatment.