Protocatechuic acid suppresses diethylnitrosamine-induced hepatic preneoplastic lesions by inhibiting phase I enzymes, reducing cell proliferation, and promoting apoptosis

Abstract

Protocatechuic acid (PCA) is a phenolic compound naturally occurring in various plants. Although numerous studies have reported on its various biological activities, information on the anti-carcinogenic potential and its molecular mechanisms in animal models has never been conclusively determined. Therefore, this study aimed to study the inhibitory effect of PCA against diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis. Rats received three intraperitoneal injections of 100 mg kg^-1 body weight of DEN to initiate hepatic preneoplastic lesions, and glutathione S-transferase placental form (GST-P)-positive foci were used as the end-point marker. Rats were treated with PCA at 40 mg kg^-1 body weight by oral gavage administration for 15 weeks to study its chemopreventive effect on the early stages of hepatocarcinogenesis. PCA treatment decreased the number and the area of hepatic GST-P-positive foci in DEN-induced rats. It inhibits the activity of cytochrome P450 reductase and reduces the expression of cytochrome P450 2E1 protein. It also suppresses cell proliferation by down-regulation of Cyclin D1 expression. Additionally, it induces apoptosis, as indicated by the up-regulation of pro-apoptotic genes, Bax and Bad, in DEN-induced rats. These findings suggest that PCA is an anti-cancer agent that inhibits hepatocarcinogenesis in DEN-treated rats.

INTRODUCTION

Liver cancer is one of the most common cancer-related deaths worldwide, accounting for approximately 8.3% of total cancer deaths (Chhikara and Parang, 2022). Chronic infection with the hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol consumption, aflatoxin exposure, etc. are factors that contribute to liver cancer development (Yang et al., 2020). Although several serums and tissues for detecting early and advanced liver cancer have been reported, the prognosis for liver cancer is still poor (Piñero et al., 2020). Nowadays, treatment options for liver cancer include surgical resection, liver transplantation, chemotherapy, immunotherapy, and radiation therapy for patients with liver cancer are outlined, but they can cause serious side effects. Many reports have shown the conclusive benefit of natural products as an alternative cancer prevention because they are relevant and promote human health without side effects (Huang et al., 2021).

In recent decades, the interest in bioactive compounds derived from edible plants has greatly increased due to their biological effects (Bishayee and Sethi, 2016; Huang et al., 2021). Phenolic acid is a major secondary plant metabolite that has pharmacological properties, including anti-cancer. Some examples of phenolic acids with anti-carcinogenic effects are gallic acid, caffeic acid, vanillic acid, and rosmarinic acid (Mirzaei et al., 2021; Punvittayagul et al., 2021; Jiang et al., 2022; Zhao et al., 2022). Although previous studies on the anti-cancer potential of phenolic acids are extensive, the discovery of new anti-carcinogenic compounds to prevent or delay carcinogenesis is still needed.

Protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) is a phenolic acid that is mostly found in medicinal herbs and our daily diet, for example, in vegetables, fruits, mushrooms, cereals, rice, and green tea. It is also a major metabolite of anthocyanins and proanthocyanins (Song et al., 2020). The molecular structure of PCA, which is rich in hydroxyl groups, enables it to act as a scavenger of free radicals. This action provides protective effects on lipids, proteins, and nucleic acids. After ingestion, PCA is rapidly absorbed in the duodenum and then undergoes methylation to form 4-methylprotocatechuic acid. This is further conjugated and transformed into O-glucuronides or O-sulphates. These conjugated forms are transported into the bloodstream via plasma proteins, where they exert their biological effects before being eliminated from the body through urine and feces (Kakkar and Bais, 2014; Song et al., 2020).

Previous studies reported its anti-inflammatory effect against lipopolysaccharide-induced kidney damage and mastitis in mice (Zhao et al., 2023; Salama et al., 2023). The anti-skin aging properties of PCA have also been reported in human dermal fibroblasts (Shin et al., 2020). Additionally, PCA treatment reduced H₂O₂-induced oxidative stress in RA fibroblast-like synoviocytes (Liu et al., 2023). The oral administration of PCA improved insulin resistance in high-fat diet-induced mice (Xiang et al., 2023). It has also been shown to have a hepatoprotective effect against menadione-induced oxidative stress in rats (Ibitoye and Ajiboye, 2020). Moreover, anti-cancer activity in ovarian and colon cancer cells was documented in previous reports (Xie et al., 2018; Acquaviva et al., 2021). However, few studies have investigated the inhibitory effect of PCA in liver cancer. Although the chemopreventive effect of PCA against diethylnitrosamine-induced rat hepatocarcinogenesis has been reported, an understanding of its molecular mechanisms underlying liver cancer development has not been fully elucidated. Therefore, the current study aimed to evaluate the underlying mechanisms of PCA in the early stages of hepatocarcinogenesis in rats induced by diethylnitrosamine.

MATERIALS AND METHODS

Animals and treatment protocol

Fifty-four male Wistar rats (4 weeks old; 90–110 g) were obtained from Nomura Siam International Co., Ltd., Thailand. They were housed in the Animal House, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand under a 12-hr light/dark cycle, 50–60% humidity at 23 ± 2°C, and allowed free access to drinking water and food. The animal protocol was approved by The Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University (Protocol No. 28/2565). All methods were performed following the relevant guidelines and regulations. This study follows the recommendations in the ARRIVE guidelines.

Our previously published article reported that PCA at 4 mg kg⁻¹ BW was an active compound in purple rice bran and presented an anticarcinogenic effect against diethylnitrosamine (DEN)-induced hepatocarcinogenesis, specifically targeting phase II detoxification and anti-inflammation (Punvittayagul et al., 2022). This study focused on understanding whether the higher dose of PCA improves its protective mechanisms against liver cancer compared to a previously tested lower dose. To explore this, a 10-time higher dose of PCA (40 mg kg^-1 BW) was administered to rats for 15 weeks to investigate the effect of concentration on cancer chemopreventive mechanisms against DEN-induced rat hepatocarcinogenesis. One week after acclimatization, the rats were randomly divided into 4 groups. Groups 1 and 2 were intraperitoneally injected with normal saline solution (4 mL kg^-1 body weight (BW). In contrast, Groups 3 and 4 were injected three times with equal volumes of 100 mg kg^-1 BW of DEN to induce hepatocarcinogenesis. In groups 1 and 3, rats orally received distilled water as negative and positive controls, respectively. In groups 2 and 4, rats were fed 40 mg kg^-1 BW of PCA to evaluate its carcinogenic and anti-carcinogenic effects, respectively. At week 5 of the experiment, six rats of each group were sacrificed to determine the inhibitory effect of PCA on the initiation stage. The remaining rats in each group were sacrificed in week 15 to evaluate their effect on the promotion stage. General observations, including body weight and water and food intake, were measured once a week throughout the experiment. The three vital organs (liver, kidney, and spleen) were excised and weighed after sacrifice to evaluate the toxic effect of the test compounds. The experimental protocol is shown in Fig. 1.

Fig. 1

Experimental procedure for carcinogenic and anti-carcinogenic properties of protocatechuic acid in rats.

Hepatic glutathione S-transferase placental form-positive foci staining

The hepatic GST-P-positive foci, the end-point marker of rat hepatocarcinogenesis, were determined by immunohistochemical staining as previously described (Thumvijit et al., 2014). The LAS Interactive Measurement program was used to measure the number and size of GST-P-positive foci as described previously (Punvittayagul et al., 2022).

Evaluation of the inhibitory effect of protocatechuic acid in the early stages of rat hepatocarcinogenesis

Phase I and Phase II xenobiotic-metabolizing enzymes

To evaluate the effect of PCA on the xenobiotic-metabolizing enzyme system, the activity of some Phase I and Phase II enzymes, including cytochrome P450 reductase (CPR), UDP-glucuronyltransferase (UGT), and glutathione S-transferase (GST), was evaluated using spectrophotometry according to our previous study (Punvittayagul et al., 2011). The expression of cytochrome P450 2E1 (CYP2E1) was determined by Western blot analysis according to our previous report (Punvittayagul et al., 2011; Punvittayagul et al., 2022). Protein expression was normalized to protein disulfide isomerase (PDI). The intensity of each band was evaluated using Image J software.

Measurement of cell proliferation and apoptosis

Double-staining immunohistochemistry was used to determine the effect of PCA on cell proliferation and apoptosis using Dako EnVision G|2 Doublestain System (Dako, Germany). Proliferating cell nuclear antigen (PCNA) was used as the end-point marker of cell proliferation. Liver sections were stained with anti-PCNA antibody (1 : 2000 dilution) and anti-GST-P antibody (1 : 1000 dilution) according to the manufacturer’s instructions. The PCNA-positive cells were examined under a light microscope as described previously (Punvittayagul et al., 2021).

The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to detect the effect of PCA on cell apoptosis. The double staining procedure was carried out according to our previous report (Thumvijit et al., 2014) and the number of apoptotic hepatocytes was counted under a light microscope following our previous study (Punvittayagul et al., 2021).

Gene expression analysis by the real-time polymerase chain reaction

The expression of some inflammation-related genes that are involved in the initiation stage of hepatocarcinogenesis was determined in the liver of rats in the 5-week protocol. The expression level of genes involved in cell proliferation and apoptosis was determined in the liver of rats in the promotion stage (15-week protocol). The total RNA was extracted and converted to cDNA as described previously. The specific primers are shown in Table 1. The PCR conditions were performed according to our previous study using a SensiFast™ SYBR Lo-ROX Kit (Bioline, France). β-actin was used as internal control and the relative fold expression of the gene was calculated by the 2^−ΔΔCT method (Punvittayagul et al., 2022).

Table 1. Primer sequences used for RT-PCR.

Gene	5′-3′ Primer sequence
TNF-α	Forward: 5′-AAA TGG GCT CCC TCT CAT CAG TTC-3′ Reverse: 5′-TCT GCT TGG TGG TTT GCT ACG AC-3′
IL-1β	Forward: 5′-CAC CTC TCA AGC AGA GCA CAG-3′ Reverse: 5′-GGG TTC CAT GGT GAA GTC AAC-3′
Cyclin D1	Forward: 5'-GTC GAG AAG AGA AAG CTC TG-3' Reverse: 5'-TTA AAA GCC TCC TGT GTG AA-3'
Bax	Forward: 5'-GTT GCC CTC TTC TAC TTT GC-3' Reverse: 5'-ATG GTC ACT GTC TGC CAT G-3'
Bad	Forward: 5'-GGA GCA TCG TTC AGC AGC AG-3' Reverse: 5'-CCA TCC CTT CAT CTT CCT CAG TC-3'
Bcl-2	Forward: 5'-CTG GTG GAC AAC ATC GCT CTG-3' Reverse: 5'-GGT CTG CTG ACC TCA CTT GTG-3'
Bcl-xl	Forward: 5'-AGG CTG GCG ATG AGT TTG AA-3' Reverse: 5'-TGA AAC GCT CCT GGC CTT TC-3'
β-Actin	Forward: 5'-ACA GGA TGC AGA AGG AGA TTA C-3' Reverse: 5'-AGA GTG AGG CCA GGA TAG A-3'

Statistical analysis

Statistical analysis was conducted using IBM SPSS Statistics Version 21. All data were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) method for comparisons among groups. Statistical significance was defined as a p-value less than 0.05.

RESULTS

Protocatechuic acid inhibits diethylnitrosamine-induced early stages of rat hepatocarcinogenesis

At 5 weeks of the experiment, the final body weight of rats treated with DEN was significantly decreased compared with the negative control group, whose final body weight was directly correlated with the decrease in food and water consumption. These findings demonstrate that DEN induces toxicity. Remarkably, PCA treatment significantly improved the final body weight and average daily water and food intake of DEN-initiated rats, indicating that PCA alleviated the toxic effects of DEN. At 15 weeks of the protocol, the results showed a recovery in body weight and water intake, and food consumption. In addition, treatment with 40 mg kg^-1 BW of PCA alone for 5 weeks significantly increased the amount of water and food consumption compared with the control group, whereas it did not lead to any change in the 15-week protocol compared with the NSS-treated group. These results showed that 40 mg kg^-1 BW of PCA had no toxicity in rats. The results are summarized in Table 2.

Table 2. General observations of rats in diethylnitrosamine-induced hepatocarcinogenesis.

Treatment	Initiation (5-week protocol)											Promotion (15-week protocol)
	n	Final body weight (g)			Food intake (g/rat)			Water intake (mL/rat)				n	Final body weight (g)			Food intake (g/rat)			Water intake (mL/rat)
NSS	6	348.8	±	9.5	23.9	±	1.3	26.3	±	1.8		5	464.0	±	9.6	22.1	±	0.4	26.7	±	2.7
NSS + PCA 40 mg kg-1 BW	6	361.3	±	17.5	27.2	±	0.9*	31.6	±	0.5*		5	453.8	±	38.6	24.0	±	2.8	25.4	±	1.6
DEN	6	280.0	±	19.6*	21.1	±	1.3*	22.4	±	0.8*		10	477.5	±	27.2	24.0	±	0.6	28.7	±	1.5
DEN + PCA 40 mg kg-1 BW	6	316.7	±	27.5**	24.4	±	0.1**	24.0	±	0.2		10	454.0	±	30.6	23.4	±	1.2	28.6	±	4.1

Values are expressed as mean ± SD.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

DEN: diethylnitrosamine, NSS: normal saline solution, PCA: protocatechuic acid, n: number of rats.

In the initiation stage (5-week protocol), the relative liver weight was significantly lower in DEN-treated rats than in control rats. In contrast, PCA significantly increased the relative liver weight of DEN-treated rats. The relative weights of the kidney and spleen did not differ significantly between the groups. During the 15-week promotion stage, PCA alone significantly reduced the relative liver weight of rats compared to the negative control group, but there was no effect on the weights of the spleen or kidney. Nevertheless, the treatment of DEN alone did not alter the sizes of the liver, spleen, and kidneys. Additionally, PCA treatment did not influence the sizes of the liver, spleen, and kidneys in DEN-initiated rats. The results are summarized in Table 3.

Table 3. Relative organ weight of rats in diethylnitrosamine-induced early stages of hepatocarcinogenesis.

Treatment		Initiation (5-week protocol)										Promotion (15-week protocol)
	n	Relative organ weight										n	Relative organ weight
		Liver			Spleen			Kidney					Liver			Spleen			Kidney
NSS	6	4.00	±	0.23	0.23	±	0.05	0.66	±	0.04		5	3.15	±	0.26	0.20	±	0.01	0.51	±	0.02
NSS + PCA 40 mg kg-1 BW	6	4.39	±	0.30	0.22	±	0.01	0.66	±	0.03		5	2.77	±	0.12*	0.18	±	0.02	0.49	±	0.03
DEN	6	3.05	±	0.17*	0.28	±	0.04	0.67	±	0.03		10	3.05	±	0.24	0.19	±	0.01	0.53	±	0.04
DEN + PCA 40 mg kg-1 BW	6	3.46	±	0.32**	0.25	±	0.02	0.67	±	0.05		10	2.99	±	0.04	0.18	±	0.01	0.52	±	0.03

Values are expressed as mean ± SD.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

DEN: diethylnitrosamine, NSS: normal saline solution, PCA: protocatechuic acid, n: number of rats.

Immunohistochemical examination of hepatic GST-P-positive foci in rats was evaluated at 15 weeks of the experiment. The results are shown in Fig. 2. There was no GST-P-positive foci formation in rats treated with PCA alone, indicating that PCA did not present carcinogenicity in the rat liver. DEN treatment significantly increased the numbers and areas of hepatic GST-P-positive foci compared to the negative control group. Interestingly, PCA treatment in DEN-induced rats significantly decreased numbers and areas of hepatic GST-P-positive foci compared to rats that received the DEN alone. These findings indicate the protective effect of PCA against DEN-induced rat hepatocarcinogenesis.

Fig. 2

Effect of protocatechuic acid on the numbers and areas of hepatic GST-P-positive foci. (A) Representative example of immunohistochemical staining for hepatic GST-P-positive foci (brown) at 15 weeks of the experiment (20×), (B) Number of hepatic GST-P-positive foci, (C) Area of hepatic GST-P-positive foci.Values are expressed as mean ± SEM, n = 5 for NSS-treated groups, n = 10 for DEN-treated groups.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

“ns” indicates not significant (p ≥ 0.05).

DEN: diethylnitrosamine, PCA: protocatechuic acid.

Inhibitory mechanisms of protocatechuic acid against diethylnitrosamine-induced early stages of rat hepatocarcinogenesis

To study the effect of PCA on the initiation stage of DEN-induced hepatocarcinogenesis, the effects of PCA on some xenobiotic-metabolizing enzymes and the expression of pro-inflammatory cytokine genes were evaluated in rat livers at 5 weeks of the experiment. To assess the activity of Phase I enzymes, we specifically examined CYP2E1, a key enzyme in DEN metabolism, as well as the activity of CPR, a microsomal electron transfer protein in the cytochrome P450 enzyme system. The results showed that the treatment of PCA alone did not alter CYP2E1 expression or CPR activity. Administration of DEN alone showed no significant effect on CYP2E1 expression but resulted in a significant increase in CPR activity compared to the negative control group. Importantly, PCA treatment led to a significant decrease in both CYP2E1 expression and CPR activity in DEN-treated rats compared to the positive control group (Fig. 3A and 3B). While PCA treatment alone significantly induced UGT activity, it did not affect GST activity compared to the negative control group. In contrast, rats treated with DEN alone exhibited significantly increased UGT and GST activities when compared to the NSS-treated rats. Unexpectedly, PCA treatment did not cause any changes in UGT and GST activities in DEN-treated rats (Fig. 3C-3D). These findings indicate that PCA at 40 mg kg^-1 BW decreases the levels of phase I enzymes involved in the biotransformation of DEN, leading to the inhibition of DEN-induced hepatocarcinogenesis. To determine the effect of PCA treatment on liver inflammation, the expression of hepatic IL-1β and TNF-α genes was evaluated. No changes were observed in the expression of IL-1β and TNF-α in rats receiving PCA alone. DEN treatment significantly up-regulated the expression levels of IL-1β and TNF-α compared to the negative control group. However, PCA treatment did not result in any changes in the expression of these genes in DEN-treated rats. The results demonstrated that PCA did not affect liver inflammation in DEN-induced hepatocarcinogenesis (Table 4). Based on these observations, PCA reduced DEN-induced hepatocarcinogenesis by inhibiting the metabolic transformation of DEN.

Fig. 3

Effect of protocatechuic acid on phase I and phase II xenobiotic-metabolizing enzymes in rat liver at 5 weeks of the experiment. (A) Cytochrome P450 2E1 protein expression. The data were normalized to the PDI protein and are expressed as the fold change relative to the negative control group, (B) Cytochrome P450 reductase activity, (C) UDP-glucuronosyltransferases activity, (D) Glutathione-S-transferase activity.

Values are expressed as mean ± SEM, n = 6.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

“ns” indicates not significant (p ≥ 0.05).

DEN: diethylnitrosamine, PCA: protocatechuic acid.

Table 4. The mRNA expression of some genes related to early stages of hepatocarcinogenesis in rats.

Treatment	Gene expression relative to β-actin (Fold change)
	TNF-a			IL-1β			Cyclin D1			Bcl-xl			Bcl-2			Bax			Bad
NSS	1.00	±	0.26	1.00	±	0.23	1.00	±	0.10	1.00	±	0.09	1.00	±	0.12	1.00	±	0.12	1.00	±	0.05
NSS + PCA 40 mg kg-1 BW	0.93	±	0.13	1.06	±	0.12	1.41	±	0.46	0.98	±	0.00	1.09	±	0.16	1.35	±	0.01*	1.60	±	0.25*
DEN	1.99	±	0.28*	2.07	±	0.26*	2.71	±	0.46*	1.11	±	0.15	1.23	±	0.11*	0.95	±	0.03	0.98	±	0.07
DEN + PCA 40 mg kg-1 BW	2.19	±	0.29	1.91	±	0.01	1.80	±	0.45**	1.02	±	0.09	1.04	±	0.14	1.26	±	0.19**	1.41	±	0.21**

Values are expressed as mean ± SD, number of rats = 6/group.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

DEN: diethylnitrosamine, NSS: normal saline solution, PCA: protocatechuic acid.

The double immunohistochemistry of proliferating cell nuclear antigen (PCNA) and GST-P was performed to determine the effect of PCA on the promotion stage of rat hepatocarcinogenesis (15-week protocol). The results are presented in Fig. 4. PCA treatment did not influence the number of PCNA-positive cells compared to the NSS-treated groups. DEN treatment significantly induced PCNA-positive cells in both GST-P-positive foci and the surrounding area compared to the NSS-treated group. Treatment with 40 mg kg^-1 BW of PCA significantly reduced the number of PCNA-positive cells in both GST-P-positive foci and the surrounding area in rats treated with DEN (Fig. 4A and 4B). The reduction in cell proliferation was also confirmed by the expression of the Cyclin D1 gene, a key regulator of cell cycle progression. As shown in Table 4, the mRNA expression of Cyclin D1 was significantly induced in the DEN alone group compared to the NSS-treated group. As expected, PCA significantly reduced the expression level of Cyclin D1 in DEN-induced rats. These findings demonstrate that PCA suppresses DEN-induced hepatocarcinogenesis by reducing cell proliferation via cyclin D1 down-regulation.

Fig. 4

Effect of protocatechuic acid on cell proliferation and apoptosis in rat liver at 15 weeks of the experiment. Arrowheads indicate stained hepatocytes. (A) Double immunohistochemical staining of GST-P (red)/PCNA (brown) and GST-P (red)/apoptotic cells (brown) in rat liver sections (20×), (B) Number of PCNA-labeled cells per 1000 hepatocytes. (C) Number of apoptotic cells per 1000 hepatocytes.Values are expressed as mean ± SD, n = 5 for NSS-treated groups, n = 10 for DEN-treated groups.

*Significantly different from the NSS-treated group, p < 0.05.

**Significantly different from the DEN-treated group, p < 0.05.

“ns” indicates not significant (p ≥ 0.05).

DEN: diethylnitrosamine, NSS: normal saline solution, PCA: protocatechuic acid.

The apoptotic DNA fragmentation in rat livers was detected using the double-labeling assay for TUNEL and GST-P, as described previously (Punvittayagul et al., 2022). Compared to the NSS-treated group, the number of apoptotic cells in the PCA alone group showed no difference. The number of hepatic GST-P-positive cells that underwent apoptosis in rats receiving DEN alone significantly increased compared to the negative control group, but it did not affect the surrounding area. As expected, PCA enhanced considerably cell apoptosis in the GST-P-positive foci in DEN-treated rats, although it did not affect the surrounding tissue. The results are presented in Fig. 4A and 4C. To examine the effect of PCA on the apoptosis pathway, the expression of pro-apoptotic genes (Bax and Bad) and anti-apoptotic genes (Bcl-xl and Bcl-2) was determined by RT-PCR. Treatment with PCA alone resulted in a remarkable induction of Bax and Bad mRNA levels in rat livers. However, it did not affect the expression of the anti-apoptotic genes, Bcl-xl and Bcl-2. Compared to the NSS-treated group, DEN significantly up-regulated the mRNA expression levels of Bcl-2 and showed a slight increase in Bcl-xl. PCA treatment significantly up-regulated the expression levels of Bax and Bad genes in DEN-induced rats, while it did not affect Bcl-xl and Bcl-2 genes compared to the positive control, as shown in Table 4. Our findings suggested that PCA could efficiently activate apoptosis in DEN-treated rats by up-regulating Bax and Bad pro-apoptotic genes.

DISCUSSION

Natural products are known to be rich in bioactive components that have pharmacological potential. One such component is PCA, a simple phenolic acid commonly found in human foods. Numerous studies have shown that PCA possesses various biological activities (Tanaka et al., 2011; Semaming et al., 2015). However, it is important to note that toxic doses of PCA have been reported. Previous studies have documented that its therapeutic dose is a low dose and that it exerts toxicity at high doses. The lethal dose (LD₅₀) of PCA in mice is 800 mg kg^-1 when administered intraperitoneally (i.p.), 3.5 g kg^-1 when given intravenously (i.v.), and 500 mg kg^-1 when taken orally (o.p.). The available literature suggests that PCA administration is safe when given at a dose of between 50 and 150 mg kg^-1, as no toxic outcomes have been reported at this range (Kakkar and Bais 2014; Song et al., 2020; Okpara et al, 2022).

Many studies have been conducted on the biological activity of PCA; however, the mechanism of its action is mostly associated with antioxidant activity. The efficacy of PCA was demonstrated in the prevention of cancers. Previous reports indicated that PCA induced cell cycle arrest and apoptosis in ovarian cancer cells (Xie et al., 2018). In addition, it also inhibited inflammation and induced apoptosis in non-small cell lung cancer (NSCLC) cell lines (Tsao et al., 2014). In 1993, Tanaka et al. reported its protective effect against DEN-induced liver cancer via alteration in hepatic ornithine decarboxylase activity (Tanaka et al., 1993). However, few reports have studied the underlying mechanisms of PCA in chemical-induced hepatocarcinogenesis.

It is well established that CYP2E1 catalyzes the activation of DEN to produce reactive electrophiles, which then react with DNA to induce mutagenesis. This metabolic process triggers oxidative stress and liver damage. However, these electrophilic intermediates are excreted from the body after detoxification by phase II biotransformation enzymes such as UGT and GST, thereby blocking the initiation stage of liver cancer development (Sindhu et al., 2013; Li and Hecht, 2022). In this study, DEN treatment significantly increased CPR activity by approximately 35%; however, it did not affect the expression of CYP2E1 protein. However, further studies are needed to assess whether DEN affects CYP2E1 enzymatic activity under these conditions. A previous study discovered that PCA within the concentration range of 0.05 to 5 mM did not inhibit the activity of CYP2E1 in acetone-induced mice (Mikstacka et al., 2002). Another study found that pretreatment with PCA at 50 mg kg^-1 resulted in a weak inhibition of hepatic CYP2E1 activity in 3-methylcholanthrene-induced rats (Krajka-Kuźniak et al., 2004). Our previous research indicated that 4 mg kg^-1 BW of PCA inhibited the activity of CPR but had no effect on CYP2E1 expression level. However, the present study showed that the administration of 40 mg kg^-1 of PCA significantly reduced both CPR activity and CYP2E1 protein expression in DEN-induced rats, indicating that PCA could block the metabolic activation of DEN. The inhibitory effect of PCA on Phase I enzymes appears to depend on concentration and experimental models.

The effect of PCA on detoxifying enzymes has been well documented. Studies have shown that treatment with 50 mg kg^-1 of PCA alone significantly increased the activity of GST, UGT, and NQO1 enzymes by 29-36% (Krajka-Kuźniak et al., 2004). PCA has been found to induce the activity of the GST enzyme through the nuclear erythroid-related factor 2 (Nrf-2) signaling pathway in menadione-induced rat liver damage and in human hepatocellular carcinoma HepG2 cells (Ibitoye and Ajiboye, 2020; Zhao et al., 2023). Our previous study revealed that administration of 4 mg kg^-1 of PCA alone resulted in a decrease in GST activity, whereas it was observed to enhance GST activity when administered to DEN-treated rats. In contrast, PCA did not affect UGT enzyme activity either given alone or to DEN-treated rats (Punvittayagul et al., 2022). It has been established that glucuronidation is the major metabolic pathway of PCA for absorption and elimination (Song et al., 2020). Although the administration of 40 mg kg^-1 of PCA alone significantly induced the activity of the UGT enzyme, it was not found to be associated with an increased activity of UGT in DEN-induced rats. It is possible that PCA may inhibit enzymes that metabolize DEN, which could result in the absence of electrophilic substances needed to activate phase II enzyme activity in DEN-treated rats.

As a result of DEN-induced liver damage, pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) are significantly elevated in DEN-treated livers, which contribute to stimulating inflammation, liver proliferation, and the development of liver cancer (Mansour et al., 2019). Numerous studies have reported the anti-inflammatory properties of PCA. For example, Akanni et al. (2020) have reported that PCA attenuated inflammation by reducing nitric oxide, TNF-α, and IL-1β in testosterone-induced benign prostatic hyperplasia rats (Akanni et al., 2020). In addition, it can reduce inflammatory mediators, including myeloperoxidase, nuclear factor kappa B (NF-κB p65), and TNF-α in LPS-induced septic lung injury in mice (Alsharif et al., 2021). Our previous report showed that 4 mg kg^-1 BW of PCA down-regulated the expression of TNF-α and IL-1β genes in DEN-induced rats (Punvittayagul et al., 2022). However, the present study found that 40 mg kg^-1 BW of PCA did not affect the levels of TNF-α and IL-1β mRNA expression in DEN-treated rats under the same experimental conditions. These findings indicate that the pharmacological actions of PCA against DEN-induced hepatocarcinogenesis differ depending on the dose used.

It is well known that the dysregulation of cell proliferation and cell death programs is related to cancer development (Loftus et al., 2022). Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA replication. The expression of PCNA is correlated with the biological activity of tumor cells (Ye et al., 2020). Therefore, immunohistochemical analysis of PCNA was performed to determine the suppressive effect of PCA on cell proliferation in DEN-induced rat hepatocarcinogenesis. Previous studies have reported that PCA suppresses oleic acid-induced vascular smooth muscle cell proliferation (Lin et al., 2015). Furthermore, it could also inhibit TGF-β1-induced cell proliferation in airway smooth muscle cells (Liu et al., 2019). In this study, we found that PCA reduced PCNA-positive cells in both hepatic GST-P-positive foci and the surrounding area, indicating that PCA suppressed cell proliferation in DEN-induced rat hepatocarcinogenesis. The mechanism of suppression was supported by the down-regulation of Cyclin D1, which is an important regulator of cell cycle progression, in DEN-treated rats. This finding revealed that PCA inhibits the expression of Cyclin D1, which would be critically important for the prevention of hepatocarcinogenesis.

Apoptosis is a type I programmed cell death that occurs when DNA damage is irreparable. Impaired apoptosis plays an important role in cancer progression (Ouyang et al., 2012). The members of the Bcl-2 family are key regulators of apoptosis and are classified into three subgroups: anti-apoptotic proteins (Bcl-2 and Bcl-xl), pro-apoptotic proteins (Bax and Bak), and pro-apoptotic BH3-only proteins (Bad and Bid) (Warren et al., 2019; Qian et al., 2022). Results of the present study revealed that PCA promotes cell apoptosis in DEN-treated rats. PCA acts as a pro-apoptotic inducer by up-regulating Bax and Bad mRNA levels in DEN-treated rats, but it did not affect the expression of the anti-apoptotic genes, Bcl-2 and Bcl-xl. These findings show that PCA induces apoptosis via the up-regulation of pro-apoptotic genes.

This study indicates that 40 mg kg^-1 BW of PCA showed cancer chemopreventive properties against DEN-induced early stages of rat hepatocarcinogenesis. The mechanism of action of PCA may be due to the inhibition of enzymes involved in DEN biotransformation, CYP2E1 and CPR. PCA also exerted anti-proliferative activity by down-regulating Cyclin D1 and inducing apoptosis via up-regulating Bax and Bad mRNA levels.

ACKNOWLEDGMENTS

This research project was supported by the CMU Junior Research Fellowship Program and the Faculty of Veterinary Medicine, Chiang Mai University. Special acknowledgment is directed to the Faculty of Veterinary Medicine and the Faculty of Medicine, Chiang Mai University for the necessary research works and instruments.

Conflict of interest

The authors declare that there is no conflict of interest.

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