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. Author manuscript; available in PMC: 2019 Aug 1.
Published in final edited form as: Toxicol Lett. 2018 Apr 22;292:31–38. doi: 10.1016/j.toxlet.2018.04.023

In vitro evaluation of structural analogs of diallyl sulfide as novel CYP2E1 inhibitors for their protective effect against xenobiotic-induced toxicity and HIV replication

Mohammad A Rahman 1, Yuqing Gong 1, Santosh Kumar 1
PMCID: PMC6015631  NIHMSID: NIHMS968127  PMID: 29694836

Abstract

Diallyl sulfide (DAS) has been shown to prevent xenobiotic (e.g. ethanol, acetaminophen) induced toxicity and disease (e.g. HIV-1) pathogenesis. DAS imparts its beneficial effect by inhibiting CYP2E1-mediated metabolism of xenobiotics, especially at high concentration. However, DAS also causes toxicity at relatively high dosages and with long exposure times. Therefore, the goal of the current study was to investigate the structural analogs of DAS for their improved toxicity profiles and their effectiveness in reducing xenobiotic-induced toxicity and HIV-1 replication. Previously, we identified commercially available analogs that possessed CYP2E1 inhibitory capacity greater than or equal to that of DAS. In this study, we evaluated the toxicity and efficacy of these analogs using hepatocytes, monocytes, and astrocytes where CYP2E1 plays an important role in xenobiotic-mediated toxicity. Our results showed that thiophene, allyl methyl sulfide, diallyl ether, and 2-prop-2-enoxyacetamide are significantly less cytotoxic than DAS in these cells. Moreover, these analogs reduced ethanol- and acetaminophen-induced toxicity in hepatocytes and HIV-1 replication in monocytes more effectively than DAS. Overall, our findings are significant in terms of using these DAS analogs as a tool in vitro and in vivo, especially to examine chronic xenobiotic-induced toxicity and disease pathogenesis that occurs through the CYP2E1 pathway.

Keywords: Cytochrome P450 2E1, Diallyl sulfide, Cytotoxicity, Alcohol, Acetaminophen, HIV-1

1. Introduction

Garlic, one of the world’s most popular condiments, has a history of use for over 7000 years. Its culinary and medicinal properties are largely attributed to its complement of organosulfur compounds. Diallyl sulfide (DAS) is one of the major organosulfur compounds found in garlic, and it has been studied extensively as a preventive/protective agent against various pathological conditions such as cancer, HIV-1, diabetes, and Parkinson’s disease (Rao et al., 2015a). DAS has attracted major attention for its inhibitory action on the cytochrome P450 2E1 (CYP2E1) enzyme and the resulting therapeutic/prophylactic benefits. CYP2E1 is a key metabolic enzyme for many xenobiotics such as alcohol and acetaminophen (Lu and Cederbaum, 2008). The inhibition of CYP2E1-mediated metabolism of these substrates, especially in overdoses, is crucially important in preventing cytotoxicity.

At low physiological concentrations, ethanol is metabolized by the enzymes alcohol dehydrogenase, catalase, and CYP2E1 (Cederbaum, 2012). However, at high concentrations, ethanol-induced CYP2E1 plays the predominant role in ethanol metabolism (Lu and Cederbaum, 2008). This results in increased production of reactive oxygen species (ROS) and other reactive metabolites, thereby causing hepatic and extra-hepatic toxicity (Ande et al., 2015; Kumar et al., 2012; Lieber, 1997; Lu and Cederbaum, 2008; Rao and Kumar, 2016a). Ethanol can also induce CYP2E1 in extrahepatic cells such as monocytes and astrocytes, causing cytotoxicity to these cells due to increased metabolism and the resulting oxidative stress. The widely-used analgesic acetaminophen (APAP), another CYP2E1 substrate, is one of the safest medications in use today. However, it can cause severe hepatotoxicity at high doses though the CYP2E1-mediated metabolism of APAP and formation of a toxic metabolite (Lee et al., 1996). APAP overdose has affected millions of people around the globe, especially in the United States, where it is the most common cause of acute liver failure (Fontana, 2008). In addition, CYP2E1 has also been found to be involved in HIV-1 replication and HIV-1/viral protein-mediated toxicity (Rao et al., 2015a). A recent study by our own group has shown that CYP2E1 can be induced by viral proteins such as gp120 (Shah et al., 2013a). It also suggests that the HIV-1 viral proteins may cause increased production of ROS and reactive metabolites, consequently increasing the viral replication, possibly through the induction of CYP2E1.

DAS is known to be a selective CYP2E1 inhibitor (Rao et al., 2015). Many in vitro and in vivo studies have been conducted using DAS either to find the mechanistic pathways of CYP2E1-associated toxic effects or to examine its efficacy as an inhibitor of CYP2E1 (Rao et al., 2015a). However, DAS itself undergoes CYP2E1-mediated metabolism and produces toxic sulfur metabolites (Brady et al., 1991a). Thus, there is a potential risk involved in using DAS as a therapeutic regimen. The challenge is to find a superior alternative to DAS with relatively low-to-negligible toxicity. We hypothesize that modification of the parent DAS structure would result in analogs with improved CYP2E1 inhibition capacity and reduced cytotoxicity. In our previous study, based on a computational ligand-docking analyses, we evaluated seven structural analogs (allyl methyl sulfide, allyl ethyl sulfide, diallyl ether, thiophene, 2-(prop-2-en-1-yloxy) ethan-1-amine, 5-hexen-1-amine, and 2-prop-2-enoxyacetamide) of DAS for their CYP2E1–inhibitory properties (Rahman et al., 2017). We found that two analogs (diallyl ether and allyl methyl sulfide) had greater CYP2E1 inhibitory capacity than DAS, while other analogs showed inhibitory capacity for CYP2E1 similar to DAS. In the current study, we used those seven analogs to evaluate their in vitro cytotoxicity and their ability to protect cells from ethanol- and APAP-mediated toxicity. We also tested these analogs for their efficacy in suppressing HIV-1 replication.

2. Materials and methods

2.1. Chemicals

The compounds tested in this study were all purchased from commercial sources. Diallyl sulfide (purity 97%), allyl methyl sulfide (AMS, purity 98%), allyl ethyl sulfide (AES, purity 98%), diallyl ether (DE, purity 98%), thiophene (TP, purity 95–98%), 2-(prop-2-en-1-yloxy) ethan-1-amine (PEA, purity 95%), and 5-hexen-1-amine (5, 1 HA, purity 95%) were purchased from Sigma-Aldrich (St. Louis, MO). 2-prop-2-enoxyacetamide (PEXA, purity 95%) was purchased from Aldlab Chemicals (Woburn, MA). The stock solutions for these compounds were prepared in dimethyl sulfoxide (DMSO).

2.2. Cell culture and treatment

For this project, we used multiple relevant cell lines: human histiocytic lymphoma U937 monocytes, SVGA immortalized astrocytes, and HepaRG hepatocytes. These cells were used because CYP2E1 is constitutively expressed and is induced by ethanol leading to cytotoxicity in these cells (Jin et al., 2011; Lu and Cederbaum, 2008; Rao and Kumar, 2016b). We also used latently HIV-infected monocytic cells (U1 cells), which is considered the model system to study the effects of HIV in monocytes (Midde et al., 2017). The U937 cell lines were obtained from ATCC (Manassas, VA). The U1 cell lines were obtained from the NIH AIDS Reagent Program (Germantown, MD). Both cell lines were cultured in Roswell Park Memorial Institute 1640 media (Sigma Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, L-glutamine, sodium bicarbonate, non-essential amino acids, and gentamycin (for U937 cells) and penicillin-streptomycin solution (for U1 cells). The SVGA astrocytes were a kind gift from Dr. Anil Kumar, University of Missouri-Kansas City (MO, USA), which were originally developed by Major et al. (Major et al., 1985). The SVGA cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with the same nutrients (except L-glutamine) as U937 media. The terminally-differentiated HepaRG cells were maintained in media prepared by the addition of the HepaRG Tox Medium Supplement® to 100 ml of William’s Medium E (ThermoFisher Scientific, Grand Island, NY) and 1 ml of GlutaMAX. All the cells were maintained in a humidified incubator with 5% CO2 at 37°C. To compare the cytotoxicity, the cells were treated with DAS and its analogs every 12 hours for 2 days (acute treatment) or 3–7 days (chronic treatment), with the addition of fresh media during each treatment after the first one. DMSO (≤0.5%) treatment served as the control condition for all experiments.

2.3 Determination of CYP2E1 activity in U937 monocytic and SVGA astrocytic cells

We demonstrated CYP2E1 enzymatic activity in U937 and SVGA cells. At first, we extracted membrane proteins and membrane-associated proteins from these cells using The Mem-PER Plus Kit following manufacturer’s protocol. Briefly, the cells were permeabilized with a mild detergent to remove the soluble cytosolic proteins. A second detergent was then added to solubilize the membrane proteins. After extraction, we quantified the protein using the Pierce BCA protein assay kit. Finally, CYP2E1 activity was determined using Vivid® CYP450 Screening Kit as described previously (Midde et al., 2016). CYP2E1 baculosomes was used as positive control for this assay. All the above mentioned kits were purchased from ThermoFisher Scientific (Grand Island, NY).

2.4. Measurement of reactive oxygen species (ROS) and cell viability using flow cytometry

To determine the level of ROS and cell viability, U937 cells were treated with the analogs, and quantification was performed using flow cytometry (BD Biosciences, San Jose, CA). We used the fluorescence dye 5-(and-6)-chloromethyl 2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Life Technologies, Oregon, USA) for ROS measurement and Ghost dye (Tonbo Biosciences, San Diego, CA) for cell viability. After 48 hours of treatment, the cells were collected and washed with PBS. The cells were resuspended in PBS containing 2.5 μl of CM-H2DCFDA and 1 μl of Ghost dye. They were incubated at 37°C without light for 30 minutes. After incubation, the cells were washed, resuspended in 300 μl of PBS, and ROS and cell viability were measured. The data were analyzed using BD FACS software (version 8).

2.5. XTT cell viability assay

Cell viability was also measured using the XTT cell viability assay kit (Cell Signaling Technology Inc., Danvers, MA). In brief, 200 μl of the media containing treated cells were collected and 50 μl of XTT detection solution was added into each well of a 96-well plate. The plate was incubated for 2–3 hours in an incubator at 37°C. The resulting absorbance, corresponding to relative viability, was measured at 450 nm using a Cytation 5 micro plate reader.

2.6. LDH activity assay

Cytotoxicity was also evaluated using the Pierce Lactate Dehydrogenase (LDH) Cytotoxicity Assay Kit (ThermoFisher Scientific, Grand Island, NY). LDH assay is a convenient method to measure cell viability in adherent cells. LDH is released into the media from damaged cells which indicates cytotoxicity and cytolysis. Briefly, 50 μl of the treated media (in triplicates from each well) were collected into a 96-well plate and mixed with 50 μl of the LDH reaction mixture. After 30 minutes of incubation at room temperature, the reaction was terminated by adding LDH stop solution. The absorbance was measured at 490 nm and 680 nm using the Cytation 5 micro plate reader.

2.7. Caspase-3 activity assay

Activation of the caspase-3 pathway is a hallmark of apoptosis. To measure caspase-3 activity in the treated cells, we used a caspase-3 colorimetric assay kit (BioVision, Inc., Milpitas, CA). After treatment, the cells were lysed using cell lysis buffer and protein was extracted. Then the protein concentration was determined using the BCA Protein Assay Kit (Pierce Biotechnology, Waltham, MA). About 50–100 μg of the protein was diluted to a final volume of 50 μl in lysis buffer. Later, 50 μl of 2X reaction buffer (containing 10 mM DTT) and 5 μl of 4 mM DEVD-pNA substrate was added and incubated at 37°C for 1 hour in darkness. The absorbance was measured by using a Cytation 5 micro plate reader at a wavelength of 405 nm.

2.8. HIV p24 ELISA

Suppression of the viral load was measured by using the p24 ELISA kit (ZeptoMetrix Corp, Buffalo, NY). The U1 cells were treated with the analogs and media was collected after 48 hours of treatment. The viral p24 group-specific antigen was specifically captured with a monoclonal antibody and then reacted with a high titered human anti-HIV-1 antibody conjugated with biotin. After incubating the sample with streptavidin-peroxidase, absorbance was measured using a micro plate reader. The OD value is proportional to the viral p24 load in the sample.

2.9. Statistical analysis

Data were analyzed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA). Data in this manuscript for all experiments are presented as mean ± SEM of 3–6 experimental replicates. One-way ANOVA was used for comparisons between different treatment groups and p-values of ≤0.05 were considered statistically significant.

3. Results and discussion

We hypothesize that the modification of the parent DAS structure will not only improve the CYP2E1 inhibitory capacity of the analogs, but will also reduce the potential toxicity at high concentrations. It is known that the sulfur hetero atom in the DAS structure interacts with the CYP2E1 active site and thereby acts as a competitive inhibitor (Brady et al., 1991b). However, this same phenomenon is also responsible for producing the toxic sulfur metabolites (Rao et al., 2015a). To find a superior alternative, we performed a structure-activity relationship study. We chose the commercially available DAS structural analogs in such a way that it would maintain its capacity to inhibit CYP2E1 while no longer being a substrate. The selected DAS analogs had either the oxygen as a hetero atom in place of sulfur (DE, PEA, and PEXA) or lacked the hetero atom completely (5,1 HA). These substitutions/deletion are likely to result in producing a nontoxic metabolite or no metabolite at all, mainly due to the absence of sulfur atom in the structure (Rao et al., 2015a). We also chose a DAS analog that had a stronger nucleophile at the carbon atom adjacent to the hetero atom (PEA, PEXA, and 5,1 HA). These alterations in DAS analogs are expected to result in improving their toxicity profiles. Furthermore, smaller side chains (AMS and AES) have been reported to increase the binding efficiency to the active site (Rao et al., 2015a). Unlike the other CYP enzymes, the binding site of the CYP2E1 lies in a narrow channel (Collom et al., 2007). Hence, a low molecular weight cyclic compound (TP) is expected to work as a potent inhibitor but not as a substrate due to its cyclic structure. Our inhibition study showed that two analogs (DE and AMS) were stronger CYP2E1 inhibitors than DAS, TP had a similar inhibitory capacity to DAS, and the other analogs showed moderate CYP2E1 inhibition (Rahman et al., 2017). Despite the differences in the magnitude of their inhibition, we included all seven analogs for this in vitro toxicity and efficacy study. We used four different cells for this project: U937 monocytes, U1 monocytes, SVGA astrocytes, and HepaRG hepatocytes.

3.1 CYP2E1 activity in U937 monocytic and SVGA astrocytic cells

To demonstrate the efficacy of the DAS analogs as CYP2E1 inhibitor, we used three CYP2E1 expressing relevant cell lines in this study. Among them, HepaRG is a well-established terminally differentiated cell lines that mimics the characteristics (e.g. CYP expression) of the primary hepatocytes. In a separate study, we have shown that CYP2E1 is expressed in high level in HepaRG cells (Kumar et al., 2017). However, CYP2E1 is expressed in lesser magnitude in monocytic and astrocytic cells compared to hepatocytes. In our previous studies, we observed substantial CYP2E1 mRNA and protein level in U937 and SVGA cells (Jin et al., 2013; Shah et al., 2013b). Since there is no report of CYP2E1 enzymatic activity in these cells, in this study, we examined CYP activity in these cells as described in Materials and Methods. We obtained around 100±10 μg of protein from 5 million U937 cells and 180±10 μg of protein from 7–8 million SVGA cells. We used various amount of protein to determine the concentration dependent increase in activity. The data showed that in both cell lines, CYP2E1 activity increased with time- and protein concentration-dependent manner (Supplementary Fig. 1A & B). CYP2E1 activity in U937 cells was comparatively higher than that in SVGA cells, which is consistent with our previous finding for CYP2E1 mRNA and protein levels in these cell lines (Jin et al., 2013). CYP2E1 baculosomes showed linear increase in activity with time (Supplementary Fig. 1C). This data justifies the use of these two cell lines for this current study, along with the HepaRG cells which resembles many characteristics of primary hepatocytes.

3.2. Effect of acute treatment of DAS analogs on cytotoxicity

At first, we conducted a time and concentration-dependent cytotoxicity study in U937 cells using the following commercially available analogs: DE, TP, AMS, and AES. We used this cell line to optimize time and concentration, because it is easily available and cost-effective compared to primary cells. We treated the U937 cells with 10–200 μM of DAS and its analogs for up to 48 hours. Using flow cytometry, we observed that DAS caused significant cytotoxicity at 50 μM, whereas DAS analogs did not show detectable toxicity (Fig. 1A). At higher concentrations, e.g. 200 μM, DAS caused severe toxicity, whereas DE, TP, and AMS did not cause significant toxicity. We also measured the level of ROS. The results showed that DAS as well as its analogs did not cause increased ROS (Fig.1B). The results suggest that DAS and its analogs do not cause cytotoxicity through the oxidative stress pathway.

Figure 1.

Figure 1

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Effect of acute treatment on cell viability and ROS production in monocytes. A) U937 cells were treated with DAS and the commercially available analogs DE, AES, AMS, and TP for 48 hours. Cell viability was quantified by flow cytometry. DAS caused severe toxicity at higher concentrations whereas DE, TP, and AMS did not show significant toxicity up to 200 μM. B) ROS production was quantified by flow cytometry. The mean fluorescent intensity (MFI) indicates the level of ROS. * indicates significance (p<0.05) compared to control, # indicates (p<0.05) compared to DAS.

We performed further investigation using three other custom-made DAS analogs: PEA, PEXA, and 5,1 HA. Since 200 μM DAS caused maximum cytotoxicity, we used only this concentration with the custom-made DAS analogs (Fig. 2A). Using the XTT cell viability assay, we observed that after 48 hours of treatment, DE, TP, AMS, and PEXA did not cause any significant cytotoxicity in monocytes, astrocytes, or hepatocytes (Fig. 2B, C, & D). The results further suggest that all the DAS analogs tested have better toxicity profiles than DAS, at least for acute treatment in both hepatic and non-hepatic monocytic and astrocytic cells.

Figure 2.

Figure 2

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Effect of acute treatment on cell viability in monocytes, astrocytes, and hepatocytes. A) The structure of the commercially available DAS analogs. B, C, and D) Cell viability in U937, SVGA, and HepaRG cells after 48 hour of treatment, as measured by XTT cell viability assay. * indicates significance (p<0.05) compared to control, # indicates (p<0.05) compared to DAS.

3.3. Effect on caspase-3 activity

It has been reported that DAS is metabolized to reactive metabolites and cause toxicity through the caspase-3-dependent apoptotic pathway (Wu et al., 2011). To determine whether the new DAS analogs show a lack of toxicity as a result of limited metabolism of these analogs into toxic species and subsequent lack of caspase-3 cleavage activation, we treated the U937 cells with 200 μM DAS analogs and measured caspase-3 activity. We observed that DAS, as expected, caused a ~2-fold increase in caspase-3 activity, whereas the DAS analogs did not cause any increase in activity (Fig. 3). The results suggest that the DAS analogs do not induce caspase-3 apoptotic pathway.

Figure 3.

Figure 3

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Effect on caspase-3 activity after treatment with DAS and its analogs. Cells were treated with 200 μM of these compounds for 48 hours and caspase-3 activity was measured by colorimetric assay. * indicates significance (p<0.05) compared to control, # indicates (p<0.05) compared to DAS.

3.4. Effect of chronic treatment on cytotoxicity with the selected DAS analogs

One of the major concerns related to the use of DAS as a research tool or potential adjuvant therapy is that it has the potential to cause toxicity over an extended period, even at relatively low concentrations. Therefore, we performed a chronic treatment study using U937 cells. We treated the cells with varying concentrations (5–200 μM) of the analogs for up to 7 days and visually examined cell viability every 24 hours under the microscope (Fig. 4A). In addition, we performed XTT cell viability assays on day 4 and day 7. The results showed that on the fourth day of treatment, all the analogs were significantly less cytotoxic than DAS at 200 μM (Fig. 4B). After seven days of treatment, the analogs showed some degree of toxicity at higher concentrations (100 and 200 μM), but still significantly less toxicity than DAS. We also treated the HepaRG hepatocytes at 200 μM DAS and its analogs. We observed that DE, TP, 5,1 HA, PEA, and PEXA did not show any toxicity after 7 days of treatment. The results suggest that the new analogs have better toxicity profiles than DAS for even longer treatment regimens.

Figure 4.

Figure 4

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Effect of chronic treatment on cell viability in monocytes and hepatocytes. U937 cells were treated with varying concentrations of DAS, TP, AMS, DE, and PEXA for 4–7 days. A) Microscopic images of the U937 cells of different treatment groups were taken on days 1, 4, and 7. B) Cell viability was quantified by XTT assay. * indicates significance (p<0.05) compared to 5 μM of the respective analog. C) Hepatocytes were treated with 200 μM of the compounds for seven days. Cell viability was quantified by XTT cell viability assay. * indicates significance (p<0.05) compared to control, # indicates (p<0.05) compared to DAS.

3.5. Protective effect of the analogs from ethanol-induced toxicity

Upon establishing the relative toxicity profiles of the novel DAS analogs using an in vitro system, we evaluated their efficacy in preventing xenobiotic-mediated toxicity and HIV-1 replication. Besides DAS being known as a selective CYP2E1 inhibitor, many studies have also shown that DAS administration induces anti-oxidant enzymes by modulating the Nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway, which plays crucial role in maintaining redox homeostasis (Ho et al., 2012; Kalayarasan et al., 2009). Moreover, DAS has been shown to increase the reduced glutathione (GSH): oxidized glutathione (GSSG) ratio, which is indicative of increased antioxidant capacity (Rao et al., 2015b). We hypothesize that due to analogous chemical structure and CYP2E1 inhibitory property, the new DAS analogs will exert their protective function by inhibiting CYP2E1 mediated metabolism as well as by inducing antioxidant capacity. At first, we used these analogs to determine whether they can rescue ethanol-induced toxicity. Among all the alcohol-related disorders, alcoholic hepatitis is considered as the most severe form of alcohol-induced liver injury (Louvet and Mathurin, 2015). Unfortunately, there is currently no effective treatment regimen available on the market. Nearly half the patients suffering from alcoholic hepatitis do not benefit from the treatment (Singal et al., 2014). Therefore, it is of critical importance to intervene the progression of ethanol-induced liver injury by taking preventive measures. CYP2E1-mediated ethanol metabolism and oxidative stress have been implicated as major contributors to alcohol-related liver injury. CYP2E1 is also induced by ethanol, which further exacerbates tissue injury (Lu and Cederbaum, 2008). Ethanol can cause damage in the case of both regular/social and binge drinking scenarios.

We co-treated hepatocytes with 50 mM ethanol (~physiological concentration in binge and/or chronic drinking) and 50 μM of the novel analogs for 48 hours. We optimized the concentration of DAS and its analogs based on our results from toxicity study, which showed that DAS was toxic at 50 μM concentration, whereas the novel analogs did not cause any significant toxicity at 50 μM. Since the objective of this study was to find a suitable alternative to DAS which can be used at higher concentration for longer duration, we used this concentration to assess whether the new analogs can protect the hepatocytes from alcohol induced toxicity. The XTT cell viability assay showed that compared with DAS, DE, TP, AMS, PEA, and PEXA rescued ethanol-induced toxicity (Fig. 5A). We also co-treated the cells with 20 mM ethanol (~physiological concentration in regular/social drinking) and 20 μM of the analogs for 48 hours, expecting that DAS would prevent ethanol-induced toxicity at a lower dose. We performed LDH cytotoxicity assay as a cytotoxicity marker for this experiment. Upon the damage of cell membrane, LDH is released into the cell culture media and indicates cytotoxicity. As expected, DAS rescued ethanol-mediated toxicity. However, TP, AMS, and PEXA showed even higher magnitudes of rescue for ethanol-induced toxicity than DAS (Fig. 5B). Overall, these results are encouraging in the search for a better alternative to DAS in preventing ethanol-induced toxicity.

Figure 5.

Figure 5

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Rescue from ethanol-induced toxicity in HepaRG hepatocytes. A) Cells were co-treated with 50 mM ethanol and 50 μM of DAS and its analogs for 48 hours. XTT cell viability assay was performed to measure cell cytotoxicity. B) Cells were co-treated with 20 mM ethanol and 20 μM of the compounds for 48 hours. LDH activity assay was performed to evaluate comparative cytotoxicity. * indicates significance (p<0.05) compared to control, £ indicates (p<0.05) compared to ethanol (ETH), # indicates (p<0.05) compared to DAS+ETH.

3.6. Protective effect of DAS analogs from acetaminophen-induced toxicity

Our next goal was to evaluate the efficacy of these DAS analogs in preventing APAP-induced toxicity. At elevated levels, CYP2E1-mediated metabolism of APAP produces the toxic metabolite-NAPQI, which potentiates liver injury. We co-treated the cells with 0.5 mM APAP and 20 μM of the analogs for 4 days. We measured the LDH activity in the media, which is indicative of enhanced cytotoxicity, every 24 hours for four days (Fig. 6). Despite DAS being a CYP2E1 inhibitor, it did not rescue APAP-mediated toxicity, but rather increased the toxicity further with APAP. However, TP, AMS, DE, PEA, and PEXA rescued APAP-induced toxicity. Furthermore, DAS showed a time-dependent increase in APAP-mediated toxicity, with severe toxicity after 4 days. However, TP, AMS, and DE showed protective effect against APAP even after 4 days of exposures. This further suggests that DAS is not an ideal CYP2E1 inhibitor to rescue APAP-mediated toxicity for chronic exposure. However, the DAS analogs do have the potential to inhibit CYP2E1 and rescue toxicity caused by chronic APAP exposure.

Figure 6.

Figure 6

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Rescue from APAP-induced toxicity in HepaRG hepatocytes. A, B, C and D) Cells were co-treated with 0.5 mM ethanol and 20 μM of DAS and its analogs for 48, 72, 96, and 120 hours. Cytotoxicity was measured by LDH activity assay. * indicates significance (p<0.05) compared to control, £ indicates (p<0.05) compared to APAP, # indicates (p<0.05) compared to DAS+APAP.

3.7. Suppression of viral p24 antigen by DAS analogs

HIV-1 viral proteins (e.g. gp120) have been implicated to increase HIV-related toxicity by inducing the CYP2E1 enzyme. Induction of CYP2E1 can result in increased oxidative stress and cytotoxicity via the metabolism of endogenous compounds, which in turn can increase viral replication (Shah et al., 2013). We treated U1 (HIV-infected U937) monocytic cells with 20 μM of the analogs. The results clearly demonstrated that the use of a CYP2E1 inhibitor can help reduce the viral load (p24) in the HIV-infected population. DAS, TP, AMS, and DE significantly lowered the viral load relative to the untreated control (Fig. 7). In addition, TP and DE showed higher viral suppression than DAS, suggesting that these DAS analogs have better capability to suppress HIV-1 replication than DAS. This is an important finding that CYP2E1 inhibitors can reduce HIV replication.

Figure 7.

Figure 7

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Suppression of viral load by DAS and its analogs in U1 cells. Viral p24 load was quantified by p24 ELISA. * indicates significance (p<0.05) compared to control.

4. Conclusion

Based on the results discussed above, we conclude that we have identified several analogs of DAS, especially TP, AMS, and DE. These compounds appear to be less toxic than DAS for the cellular models evaluated in this project. This is the first report of an extensive in vitro toxicity profile study of DAS and its analogs using hepatic and extra-hepatic cells. Our next goal is to evaluate the pharmacokinetic and pharmacodynamic properties of these DAS analogs using an appropriate animal model. Upon further analysis, these analogs may replace DAS as a research tool and/or potential therapeutic regimen for preventing xenobiotic-induced toxicity and viral suppression. Despite having many therapeutic properties, the usage of DAS is restricted due to its toxicity. Therefore, there was a critical need for replacing DAS with a safer CYP2E1 inhibitor.

Supplementary Material

1

Acknowledgments

The authors thank the National Institutes of Health for financial support to Dr. Kumar (AA022063). The authors also acknowledge Regional Biocontainment Laboratory for providing access to work with HIV-1-infected cells, and Molecular Bioscience Resources for performing oxidative stress study using Floy Cytometry.

Abbreviations

CYP

Cytochrome P450

APAP

Acetaminophen

DAS

diallyl sulphide

AMS

allyl methyl sulphide

AES

allyl ethyl sulphide

DE

diallyl ether

TP

thiophene

PEA

2-(prop-2-en-1-yloxy) ethan-1-amine

5,1 HA

5-hexen-1-amine

PEXA

2-prop-2-enoxyacetamide

ROS

Reactive oxygen species

Footnotes

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Conflict of interest

The authors declare no conflict of interest.

Authorship contributions

Participated in research design: Mohammad A. Rahman and Santosh Kumar

Conducted experiments: Mohammad A. Rahman and Yuqing Gong

Performed data analysis: Mohammad A. Rahman and Santosh Kumar

Wrote the manuscript: Mohammad A. Rahman and Santosh Kumar

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