2017 Volume 40 Issue 9 Pages 1590-1594
A wide range of medications are routinely used to maintain and improve human health. Hence, it is essential that we understand and predict adverse effects caused by the combined use of multiple medications. In the present study, we investigated whether the combination of carbon tetrachloride (CCl4) and acetaminophen (APAP) had a detrimental effect on the liver. Mice injected with APAP (100 mg/kg) showed no significant changes in hepatic injury markers (alanine aminotransferase and aspartate aminotransferase), histopathological findings, pro-inflammatory cytokine levels, or hepatic oxidative stress. In contrast, a single injection of CCl4 (15 mg/kg) led to a significant increase in hepatic injury, in addition to an increase in pro-inflammatory cytokine levels and oxidative stress. Co-administration of APAP and CCl4 resulted in exacerbation of these hepatic injuries. Our results suggest that a non-toxic dose of APAP has the potential to increase CCl4-induced liver damage and oxidative stress.
In the modern world, many types of medicines are used routinely. In Japan, approximately 700 million medicines are prescribed each year (2017, the Ministry of Health, Labour and Welfare of Japan). Although each medication may be safe when administered individually, the combined use of multiple drugs increases the possibility of adverse effects. For example, the interaction of phenobarbital and warfarin is critical, since phenobarbital induces CYP and enhances warfarin de-activation.1) Although drug interactions are already reported in package inserts, these are not exhaustive. In addition, new drugs are constantly being developed and sold. Hence, it is essential to understand and predict any additive effect caused by drug combinations.
The liver plays important roles in the detoxification of harmful compounds and in homeostasis, and is the main compound-secreting organ.2) Drug metabolism primarily occurs in the liver,3) and the toxic effects of drugs, chemicals, and their metabolites manifest primarily in the liver.
Carbon tetrachloride (CCl4) is a well-known toxic chemical used to induce hepatic injury in a wide range of laboratory animals.4) CCl4 causes lipid oxidation and oxidative stress through CYP2e1-mediated generation of highly reactive radicals, resulting in severe hepatocellular necrosis 5. Acetaminophen (APAP) is a popular analgesic and antipyretic drug at therapeutic doses, but is also well-documented to cause liver injury.6) Indeed, the main cause of liver failure in industrialized countries is APAP overdose. By means of this effect, APAP is also used as a hepatotoxic chemical in animal models.7) APAP-induced liver injury is initiated by the formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is generated through the action of CYP2e1. NAPQI depletes glutathione (GSH) and covalently binds to proteins. Consequently, oxidative stress increases, leading to hepatic necrosis.8)
In recent years, research into the additive effects of APAP and deltamethrin, cadmium, angiotensin-converting enzyme inhibitors, and eplerenone on the liver have been reported.9,10) In addition, the combined effects of isoniazid and rifampicin,11) alcohol and methotrexate,12) and disulfiram and APAP13) on the liver have been studied. These studies mainly focused on interactions due to CYP induction/inhibition. To the best of our knowledge, related actions, which induce hepatic injury in both, have not been reported. Therefore, we investigated whether co-administration of CCl4 and APAP resulted in additive negative effects on the liver.
CCl4 solution, polyethylene glycol (PEG), olive oil, 15% formalin neutral buffer solution (6% formalin, pH 7.4: formalin), xylene, ethanol, and acetic acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). APAP was obtained from Yoshida Pharm Co. (Saitama, Japan) and was dissolved in 50% PEG and 50% saline (vehicle). CCl4 was diluted in olive oil. All other reagents and solvents were of analytical grade.
Male six-week-old ddY mice (28–30 g) were purchased from Japan SLC (Shizuoka, Japan), and were maintained under standard conditions of controlled temperature (24±1°C), humidity (55±5%), and light (8:00/20:00 light/dark cycles) with free access to water and food. The mice were acclimatized under laboratory conditions for 1 week, and seven-week-old mice were used for experiments. Following the experiments, all mice were killed using pentobarbital. All experiments were approved by the Institutional Animal Care and Experiment Committee of Kinjo Gakuin University (No. 129).
Mice were randomly divided into four groups of nine or ten. Mice in group 2 (APAP group: n=10) and group 4 (APAP+CCl4 group: n=9) received 100 mg/kg (5 mL/kg) APAP via intraperitoneally (i.p.) injection at 18:00. Mice in group 1 (control group: n=9) and group 3 (CCl4 group: n=10) were injected (i.p.) with equivalent volumes of vehicle at 18:00. Thirty minutes after the APAP or vehicle injection, both the CCl4 group and APAP+CCl4 group mice were injected (i.p.) with 15 mg/kg (5 mL/kg) CCl4. The control and APAP group mice were injected (i.p). at 18:30 with equivalent volumes of olive oil. Sixteen hours after the final administration, mice from each group were euthanized and bled to obtain plasma. Plasma was stored at −80°C. The livers were rapidly removed and snap frozen in liquid nitrogen and subsequently stored at −80°C or fixed in formalin solution for histological assay. Experimental procedure of figure is described in Fig. 1.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using the Transaminase CII Test (Wako Pure Chemical Industries, Ltd.) according to the manufacturer’s instructions and as previously described.14,15) Plasma levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instructions (eBioscience, San Diego, CA, U.S.A.). For relative quantification, calibration curves were prepared using standard solutions.
A portion of left liver from each mouse was fixed in formalin solution, dehydrated, and embedded in paraffin. Sections of 4 µm thickness were obtained from the paraffin-embedded blocks. These sections were dewaxed in xylene and rehydrated in a graded ethanol series. After rehydration, sections were stained with Mayer’s hematoxylin solution (Nacalai Tesque, Kyoto, Japan). After rinsing in running tap water, these were stained with 0.1% eosin solution (Wako Pure Chemical Industries, Ltd.) containing acetic acid. Finally, the sections were dehydrated, cleared, and mounted with cover glass. Histopathological features of each slice were examined under a light microscope.
Total MDA levels in the liver were examined via a colorimetric microplate assay (Oxford Biochemical Research, Oxford, MI, U.S.A.) according to the manufacturer’s protocol and as previously described.16)
Hepatic GSH levels were measured using a GSSG/GSH quantification kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions and as previously described.17)
Total RNA was extracted from 0.1 g liver sections using the ISOGEN II kit (Nippon Gene, Tokyo, Japan). qRT-PCR was performed with the One-Step SYBR PrimeScript PLUS RT-PCR kit (Perfect Real Time) (TaKaRa Bio, Shiga, Japan) using an Applied Biosystems 7300 system (Applied Biosystems, Foster City, CA, U.S.A.). PCR conditions and primers were as previously described.18) The amount of each target mRNA quantified was normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding mRNA.
Statistical analyses of the multiple comparisons were performed via one-way ANOVA with a post-hoc Tukey–Kramer’s test. All statistical analyses were performed using SPSS 19.0 software (Chicago, IL, U.S.A.). Values of p<0.05 were considered statistically significant.
First, we analyzed plasma levels of ALT and AST (Fig. 2), which are recognized markers of hepatic injury and dysfunction. Control and APAP groups showed normal ALT (Fig. 2A) and AST (Fig. 2B) levels. Administration of CCl4 led to an increase in ALT and AST plasma levels. In addition, co-administration of APAP and CCl4 increased ALT and AST levels more than CCl4 alone did.
Male ddY mice were injected (i.p.) with 100 mg/kg APAP at 18:00. Thirty minutes later, the mice were injected (i.p.) with 15 mg/kg CCl4. ALT (A) and AST (B) levels were determined from plasma samples 16 h after the second injection. Data are presented as mean±S.D. of 9 or 10 mice. ** p<0.01 versus control group. ## p<0.01 versus CCl4 group.
Simultaneously, we evaluated histopathological changes in the liver tissue. Liver sections stained with hematoxyline–eosin (H&E) showed normal cell morphology, well-preserved cytoplasm, and clear, plump nuclei in the control and APAP-treated groups. In contrast, necrosis was observed around the central vein (zone 3) in CCl4-injected mice (Figs. 3C, 3G). This necrotic area increased in the APAP+CCl4 group (Figs. 3D, H).
Mice were treated as described in the legend for Fig. 2. Liver specimens were stained with H&E. Micrographs provide representative liver sections obtained from the control (A and D), APAP (B and E), CCl4 (C and F), and APAP+CCl4 (D and G) groups. Black arrows indicate necrotic areas and bars indicate 0.15 mm.
Next, we measured plasma TNFα and IL-6 as representative inflammatory markers (Fig. 4) since exposure to APAP and CCl4 is known to elevate levels of these cytokines.5,8) Our results showed that both APAP and CCl4 slightly upregulated TNFα (Fig. 4A) and IL-6 (Fig. 4B) levels, and that levels of both significantly increased after combined treatment with APAP+CCl4.
Mice were treated as described in the legend for Fig. 2. Plasma TNF-α and IL-6 levels were determined 16 h after the second injection. Data are presented as mean±S.D. of 9 or 10 mice. * p<0.05, ** p<0.01 versus control group.
In addition to plasma biochemical analysis, we quantified hepatic MDA, which is a well-known marker of lipid peroxidation.19) Although, individually, CCl4 or APAP administration did not significantly change MDA levels, co-administration of APAP and CCl4 did increase them (Fig. 5A). Furthermore, we measured hepatic GSH levels, since GSH is critical in detoxifying chemicals.20) Although APAP administration resulted in no significant change, CCl4 administration induced significant decreases in GSH (Fig. 5B). Moreover, co-administration of APAP and CCl4 decreased hepatic GSH levels more significantly than CCl4 alone did. There findings suggest an additive effect of APAP and CCl4 not only on hepatic MDA levels but also on GSH levels.
Finally, we determined hepatic CYP2e1 mRNA levels (Fig. 5C). Although the CCl4 and APAP+CCl4 groups had lower levels than the control and APAP groups did, these decreases were not significant.
Mice were treated as described in the legend for Fig. 1. MDA (A), GSH (B), and CYP2e1 mRNA levels (C) in the liver were determined 16 h after the second injection. Data are presented as mean±S.D. of 9 or 10 mice. * p<0.05, ** p<0.01 versus control group, # p<0.05 versus CCl4 group.
APAP- and CCl4-induced hepatotoxicity is known to occur via a multifactorial process.5,8) The first step in this process is the metabolism of APAP or CCl4 by CYP, which converts APAP to NAPQI, and CCl4 to trichloromethyl and trichloromethyl peroxy radicals. These metabolites are then scavenged by antioxidant enzymes or react with sulfhydryl groups and protein thiols. The third step involves increased oxidative stress due to overexpression of NAPQI or free radicals, and is associated with alterations in calcium homeostasis and the initiation of signal transduction responses. The fourth step is ATP depletion and increasing cellular calcium levels, along with an inflammatory response. All these changes lead to the fifth step of cellular necrosis. Although APAP and CCl4 cause hepatotoxicity via a similar route, the mechanisms through which they cause oxidative stress differ.21) Therefore, we investigated whether the two compounds together cause an additive or synergistic effect. Specifically, CCl4 was selected as an agent that does not induce GSH depletion, while APAP was selected as a prototypical GSH-depleting agent.
Multiple compounds have been reported to protect against or exacerbate APAP- or CCl4-induced hepatotoxicity, and associated mechanisms involving the multiple steps outlined above have been proposed. In particular, several studies have reported that pre-treatment with phenobarbital, acarbose, or natural products such as Salvia officinalis, may potentiate the CYP2e1-mediated hepatotoxicity of CCl422–24). Potentiation of APAP-induced hepatotoxicity by alcohol or natural products such as Ginkgo biloba have also been reported.25,26) These interactions are based on CYP induction (the first step). Retinol, however, potentiates APAP-induced hepatotoxicity by reducing hepatic GSH levels, affecting the second and third steps.27) Our current study showed that a combination of APAP and CCl4 increased the area of necrosis (the fifth step), pro-inflammatory cytokine levels (the fourth step), and oxidative stress (the third step). In addition, CYP2e1 levels were unchanged between the CCl4 and APAP+CCl4 groups (the first step) although these decreased in comparison with control levels. These results suggest that any additive/synergistic effects occur in the second or third steps.
APAP is recognized as a safe and effective analgesic at therapeutic doses. However, an overdose of APAP can cause severe hepatic injury in humans and animals.28) In fact, the main cause of acute liver failure in industrialized countries is APAP overdose. In the present study, the dose of APAP used (100 mg/kg) was slightly higher than the highest recommended dose in humans. In Japan, this recommended dose is 4000 mg, which is equivalent to 67 mg/kg, assuming a body weight of 60 kg. Although APAP itself was not hepatotoxic at 100 mg/kg, additive or synergistic liver injury was observed when combined with CCl4-induced mild hepatic damage. In fact, co-administration of 100 mg/kg APAP and 15 mg/kg CCl4 had an effect equal to that of 20 mg/kg CCl4 on ALT and/or AST activities (data not shown). Although it is not possible to determine at this time whether this effect is additive or synergistic, these data imply that non-toxic doses of APAP might also induce severe hepatotoxicity in humans when taken with other hepatotoxic compounds. Further investigation is needed to understand this phenomenon.
In conclusion, we demonstrated that a non-toxic dose of APAP potentiates CCl4-induced hepatotoxicity in an additive or synergistic manner. To our knowledge, this is the first report suggesting that a non-toxic level of APAP can induce hepatotoxicity. Although further investigation is needed to clarify the mechanism of interaction between APAP and CCl4, these findings provide an important basis for understanding the risk of interaction and to predict an additive effect in combined drug use.
The authors thank Dr. Nobuyuki Fukuishi and Yasuro Shinohara (Kinjo Gakuin University, Japan) for his kind suggestions.
The authors declare no conflicts of interest.