Long-term oral administration of Paederia foetida decreases cytochrome P450 mRNA expression: The predictive approaches in a rat model

Paederia foetida (Linn.) is one of the medicinal plants containing various bioactive compounds (e.g., beta-sitosterol and ursolic acid), which has been widely used in medicine and cuisine among different communities. It exhibits several pharmacological properties, including anti-inflammatory, antioxidant, antidiabetic, and anticancer. However, its toxicity on the liver and kidney, as well as its effect on hepatic drug-metabolizing enzymes, lacks documentation. Therefore, the toxic effects of P. foetida on the liver and kidney and metabolizing enzymes were explored. In a recent study, male Wistar rats were grouped into five groups and gavaged daily with 0–1,000 mg/kg BW of a P. foetida extract (PFE) for 8 weeks. After euthanasia, liver, kidney, and blood samples were collected and subjected to histological and biochemical analysis. The important cytochrome P450 mainly expressed in the rat liver, including Cyp3a1, Cyp2d1, and Cyp2c6 , was investigated. There were no changes in body and organ weight between groups. However, the liver weight of rats treated with 1,000 mg/kg BW PFE was increased when compared to the control group. The liver and kidney histopathology were observed with no difference between groups. The markers of liver damage (alanine aminotransferase and aspartate aminotransferase) and kidney damage (blood urea nitrogen and creatinine) between all groups showed no change. Cyp3a1 , Cyp2d1 , and Cyp2c6 mRNA expression levels among PFE-treated groups were decreased in a dose-dependent manner, suggesting that subchronic exposure to this extract significantly reduces Cyp3a1 , Cyp2d1 , and Cyp2c6 expressions in rat livers without toxicity or change in histology and biochemical data. However, its concomitant use with prescription drugs needs further investigation.


INTRODUCTION
Paederia foetida (Linn.) (Rubiaceae family) has been used in Thai cuisine and Thai traditional medicine for diseases, including rheumatoid arthritis (Dixit, 2013;Kumar et al., 2015), diarrhea (Afroz et al., 2006), asthma (Macwan, 2010), and relieving diabetes (Kumar, 2014), in the southern region including Thailand. Phytochemical analysis revealed that P. foetida is rich in lupeol, ursolic acid, and beta-sitosterol (Dwivedi et al., 2018), which are shown to have their biological activities, including antiinflammatory (Das et al., 2012), antioxidant (Upadhyaya, 2013), antidiabetic (Morshed et al., 2012), and anticancer (Reddy et al., 2011) activities. Although the use of this plant is widespread, information about its toxicity-related issues is still limited (Reddy et al., 2011). Moreover, the long-term use of herbal remedies has also been associated with liver toxicity (Teschke et al., 2013), adverse effects on kidney function (Amadi and Orisakwe, 2018), and herb-drug interaction (Hu et al., 2005). To better understand P. foetida use and toxicity-related issues, it is important to promote awareness of possible health complications and promote the safety of long-term effects of P. foetida accumulation in rat organs and toxicity associated with herbs (Turkmenoglu et al., 2016).
After xenobiotics enter the human body, they undergo further metabolism (biotransformation), mainly in the liver. In the liver, xenobiotic sensors, e.g., pregnane X receptor (PXR) and constitutive androstane receptor (CAR), coordinate the transcriptional control of the P450-metabolizing enzyme, which catalyze rate-limiting steps in xenobiotic clearance or biotransformation (Chai et al., 2016;Chang et al., 2017). Moreover, more than 75% of pharmaceutical drugs undergo first-pass metabolism in the liver by cytochrome p450 enzymes (Wienkers and Heath, 2005). Therefore, the biotransformation of xenobiotics was expected to alter the function of drug-metabolizing P450 enzymes, leading to therapeutic failure or the activation of toxicity mechanisms. In previous reports, herbal plants were shown to cause in vitro CYP inhibition and upregulation and downregulation of CYP mRNA expression, resulting in toxic effects (Gravel et al., 2018). However, the impact of the P. foetida ethanol extract on in vivo mRNA expression was less well documented.
There is no published report on the effect of the P. foetida ethanol extract on the subchronic toxicity and CYP mRNA expression in animal models. Therefore, this study aimed to investigate the effects of P. foetida on i) subchronic toxicity concerning damage to the liver and kidney function and ii) the alteration of the mRNA expression level of hepatic phase I drug-metabolizing enzymes in rats. To predict and avoid herbdrug interaction, the predominant forms of cytochrome p450 in rat livers, including Cyp3a1, Cyp2d1, and Cyp2c6, have been considered in this study (Martignoni et al., 2006).

Plant materials
Paederia foetida leaves (voucher sample: Ref. HA1601061505) were obtained from Pak Phanang, Nakhon Si Thammarat, Thailand (8°17′54.5″N 100°09′13.1″E). Paederia foetida leaves were cut into small pieces and subjected to a hot air oven for 48 hours at a temperature of 60°C. Paederia foetida leaves powder was soaked in 95% ethanol for 3 days (three times) at the ratio of a 100 g sample: 1 L ethanol. After ethanol evaporation, a crude semisolid residue was obtained with a percentage yield value of 20 /100 g (dried P. foetida leaf powder).

High Performance Liquid Chromatography (HPLC) and mass analysis
100 µg of the P. foetida leaves ethanol extract was subjected to HPLC.. The separation was performed using a reverse phase C18 column as the stationary phase, whereas the mobile phase contained water (A) and acetonitrile (B). The peak chromatogram of the P. foetida leaves ethanol extract was eluted by the condition which was set as the following: 0-30 minutes, 25%-100% B; 30-45 minutes, 100% B; 45-50 minutes, 100%-25% B; 50-60 minutes, 25% B. The major peak eluted at 12.48 minutes was collected and subjected to a mass analyzer (directed methods). Mass per charge ratio was employed by electrospray ionization (ESI) (Bruker Daltonics GmbH, Bremen, Germany). The mass spectrometry (MS) spectra were acquired in positive modes, and m/z were scanned in the range of 100-1,000 and acquired by HyStar (V3.2). Parameters for MS were as follows: nebulizer gas (nitrogen): 9 l/minute, spray voltage: 4,000 V, ESI soured temperature: 365°C, and collision gas (helium) pressure: 9.6 × 10 −6 Torr. Bruker Daltonics Data analysis version 3.1 was used for ion chromatogram extraction.

Animals
The experimental animals (7-week-old male Wistar rats weighing 170-200 g) in this study were obtained from the National Laboratory Animal Center, Nakhon Pathom, Thailand. After arrival at the Walailak Animal Center, the animals were acclimatized before the experiment for 5 days and housed under controlled conditions of 22°C ± 2°C, 55% ± 10% humidity, and a 12:12 hours light: dark cycle with food and water ad libitum. The research protocol for the animal study was approved by the Institutional Animal Care and Use Committee, Walailak University (WU-AICUC-63008).

Experimental design
After the acclimation period, rats were randomly assigned into five groups (n = 6). Each group was treated daily (p.o.) with either 0.25% CMC for the control group or 50, 100, 500, and 1,000 mg/kg PFE for the treated groups. Moreover, the rats were checked once a day for signs or symptoms of toxicity including illness, injury, and abnormal behavior. After 8 weeks, a gross necropsy and complete gross postmortem examinations were performed after all animals were euthanized. Organs, including the brain, spleen, liver, and kidneys, were collected and weighed to further calculate absolute and relative organ weights (organ weight (g)/body weight (g) × 100). Blood samples were collected and subjected to blood biochemistry parameter analysis. The liver and kidneys were fixed in 10% neutral buffered formaldehyde for further histological analysis, and a part of the tissues was stored at −80°C after being snapfrozen in liquid nitrogen.

Hematology and biochemical studies
At the end of the experiment, rats were intraperitoneally injected with Zoletil 100 and xylazine at the ratio of 25:5 mg/kg BW (Machado et al., 2009). After rats were deeply anesthetized, blood samples were collected by a cardiac puncture and kept in ethylenediamine tetraacetic acid tubes. Complete blood count, including i) red blood cell (RBC) count, ii) white blood cell (WBC) count, iii) hemoglobin (Hb) concentration, iv) % hematocrit (Hct), as well as RBC indices including v) mean corpuscular volume (MCV), vi) mean corpuscular hemoglobin (MCH), and vii) mean corpuscular hemoglobin concentration (MCHC), was determined by the Auto Hematology Analyzer (Aspen Diagnostics, India). For other biochemical studies, serum was collected by centrifugation at 3,000 rpm for 30 minutes. After that, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine were determined by using an automated analyzer (Mindray BS-400 Chemistry Analyzer, Aspen Diagnostics, India).

Histopathological study
After blood collection, the liver and kidneys were excised and fixed in 10% buffered formalin. Liver and kidney tissue were embedded in paraffin wax and were cut at 5 µm thick sections. The histology of the liver and kidneys was determined using staining with hematoxylin and eosin dye. The histological slides and representative photomicrographs were examined and captured under a light microscope.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)
The RNeasy Mini Kit (QIAGEN, Germantown, MD) was used to extract mRNA, and 1 µg of total mRNA was then converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The specific primers for Cyp3a23/3a1, Cyp2d1, and Cyp2c6 were purchased from Applied Biosystems (Foster City, CA): Cyp3a23/3a1 (Rn01640761_gH), Cyp2d1 (Rn01775090_mH), Cyp2c6 (Rn02329961_mH), and Gapdh (Rn01775763_g1). Gene expression of target genes was amplified by real-time PCR reactions in triplicate in an ABI 7500 Sequence Detection System (Applied Biosystems). The expression of Cyp3a23/3a1, Cyp2d1, and Cyp2c6 genes was normalized to Gapdh, and quantification of relative expression was determined by Pfaffl's method (Pfaffl, 2001).

Statistical analysis
Data were analyzed using the Prism 9 software (GraphPad, La Jolla, CA) and presented as means ± SD. Mean differences between groups were performed using a two-tailed unpaired Student's t-test or one-way analysis of variance. Significant differences were considered when the p-value was less than 0.05.

HPLC analysis and PFE
We preliminarily determined the constituents of the P. foetida ethanol extract (PFE); 100 µg of the crude ethanol extract of the P. foetida ethanol extract was subjected to the HPLC and mass analyzer. The major peak of the P. foetida ethanol extract under our condition was eluted at 12.48 minutes (Fig. 1A). According to the mass analyzer, 2 ion mass [M-H] + = 413 (Fig. 1B) and [M+H] + = 457 (Fig. 1C) were observed and identified as beta-sitosterol (Rozenberg et al., 2003) and ursolic acid (Novotny et al., 2003), which are consistent with the previous report, respectively.

Changes in body weight and organ weight
To determine the toxic effect of P. foetida, rats were treated with different doses of PFE (0-1,000 mg/kg). The change in body and organ weight was firstly determined as an indicator of toxic effects (Lazic et al., 2020). The results showed that the change in body weight among the treated group was not observed up to 1,000 mg/kg/day compared to the control group (Table 1), and there was no sign of toxicity and morbidity. Moreover, organ and relative organ weights (Table 2), including brain, spleen, and kidney weight, were not significantly different between the treatment and control groups. However, the liver weight and relative liver weight of the 1,000 mg/kg PFE-treated group were slightly increased compared to the control groups. The results indicate that subchronic effects of PFE have no potential toxic effects on the body and organ weight up to 1,000 mg/kg body weight.

Hematology parameters
We next determined the effect of the P. foetida ethanol extract on hematology parameters which is a sensitive index for vital toxicity (s). In Table 3, there was no significant change in hematological values, including RBC count, Hb, Hct, platelet, and WBC count (e.g., neutrophils, lymphocytes), in the rats treated with PFE compared with the control group. This result suggests that the P. foetida constituents in the ethanol extract had a low toxic effect on the hematologic system, which was basically in the normal reference range.

Effect of PFE on liver and kidney functions
To investigate the effect of PFE on the liver and kidneys, which play important roles in xenobiotic metabolism and clearance (Woldemeskel, 2017), histology and biochemistry parameters were determined. As shown in Figures 2 and 3, no lesions were found upon macroscopic and microscopic examinations in either the liver or kidney tissue of any of the rats treated with P. foetida, as the cellular integrity and the cellular architecture were intact in both tissues compared to controls. Aside from the gross appearance and histology of the liver and kidneys, the biomarkers reflecting liver (serum ALT and AST) and kidney (serum BUN and creatinine) damage were then determined. The results revealed that all biochemical parameters for liver and kidney damage were within physiological ranges and were not significantly different in the PFE-treated group and control group, suggesting that subchronic administration of PFE might have no nephrotoxic and hepatotoxic effects.

In vivo effect of PFE on major cytochrome P450 mRNA expression
Paederia foetida was previously reported to inhibit in vitro cytochrome P450 activity (Dubey et al., 2017). However, altered expression of major cytochrome P450 enzymes in vivo is still unreported. Therefore, mRNA expressions of Cyp3a1, Cyp2c6, and Cyp2d1 in the rat liver of each treated group were determined using target-specific primers in real-time PCR. As shown in Figure 4, at 100 mg/kg of treatment, mRNA expression levels of Cyp3a1 and Cyp2d1 were 2-fold lower than that observed in the control group. However, mRNA expression levels of Cyp2c6 showed a significant decrease from that observed in the control group at 500 and 1,000 mg/kg. Therefore, the administration of P. foetida decreases mRNA expression levels of Cyp3a1, Cyp2d1, and Cyp2c6 in a dose-dependent manner, suggesting that P. foetida may interfere with xenosensor and cytochrome P450 activity.

DISCUSSION
The use of herbal supplements, both individually and in combination with prescription drugs, is continuing to increase among the global population. The naturally occurring phytochemicals in plants are abundant sources of therapeutic agents for medicinal purposes in developing countries. Although plants show the benefit needed in health maintenance, the available evidence revealed that people are frequently exposed  to various forms of toxic plants. To explore the safety use of P. foetida, Wistar rats were given daily oral administration with PFE (0, 50, 100, 500, and 1,000 mg/kg BW) for 8 weeks. The changes in body weight, organ weights, relative organ weight, all hematological and biochemical parameters, and liver and kidney histology were investigated. There was also no adverse change in the kidneys of the PFE-treated group compared to the control group. Loss of body and organ weight of more than 10% is used as an indicator of adverse side effects of toxic agent exposure (Woldemeskel, 2017). Surprisingly, 8 weeks of PFE treatment showed no body weight change between the groups, and there was no sign of toxicity and morbidity. However, we found that the mean liver weight and relative liver weight were slightly increased at 1,000 mg/kg BW of the PFE-treated group, while the abnormalities (e.g., histology, biochemistry parameters for liver damage) were not observed (Fig. 2). This result is in line with our liver function biomarker results including ALT and AST, which indicate that the liver increased in size without toxicity (Olayode et al., 2020). The increased liver weight could be due to a functional adaptation of the liver for maintaining the homeostasis toward xenobiotics by increasing the hepatocellular protein involved in hepatocellular drug metabolism such as drug transporters, thus enhancing the rate of metabolism and excretion of the xenobiotics (Xu et al., 2005). In addition, most drugs or xenobiotics and their metabolites are excreted by the kidneys. Increased levels of BUN and creatinine are the hallmarks of renal failure. However, the BUN and creatinine levels in this study showed no significant changes. There was also no adverse change in the kidneys of the PFE-treated group compared to the control group. Our results were aligned with those from previous reports that demonstrated the hepato-and nephroprotection of P. foetida in rats, including the following: i) the administration of 500 mg/ kg (p.o.) P. foetida methanal extract for 28 days protects the rat's  kidneys from alloxan-induced diabetic renal injury (Borgohain et al., 2017); ii) 200 mg/kg BW P. foetida ethanal extract has been shown to have a protective effect against paracetamol-induced acute liver injury (Roy et al., 2017); and iii) preadministration of 400-500 mg/kg BW P. foetida ethanol extract for 21 days protects the rat liver from CCl 4 -induced liver injury (Kumar, 2014;Uddin et al., 2011). Several reports, including those from our study, suggest that 100-1,000 mg/kg BW of P. foetida intake might be a safe in vivo model. The concentration of 1,000 mg/kg BW of PFE in rats can be calculated to estimate the human equivalent dose (HED) by using HED (mg/kg) = animal dose (mg/kg) × km ratio, in which the km ratio is obtained from dividing 6 (animal km factor) by 37 (human km factor), which is 162 mg/kg BW. Our calculation suggests that this dose could be used in humans without any adverse effects because of its nontoxicity to the liver and kidney (Nair and Jacob, 2016).
Herein, we reported that the first-time oral administration of P. foetida decreased the mRNA expression levels of Cyp3a1, Cyp2d1, and Cyp2c6 which play an important role in phase I metabolism in liver tissues of Wistar rats that received different concentrations of PFEs in a dose-dependent manner (Fig. 4). This may be due to the ability of the naturally occurring compounds in P. foetida to decrease the basal transcription of Cyp3a1, Cyp2d1, and Cyp2c6 mRNA. In general, mRNA expression levels of CYP-metabolizing enzymes are regulated by xenobiotic sensors acting as nuclear transcription factors (e.g., PXR and CAR). Upon binding to xenobiotic responsive elements, the CYP genes turn on, and proteins are synthesized, leading to increased first-pass metabolism of xenobiotics followed by elimination from the body. We suppose that the major compounds found in the P. foetida extract (PFE) (beta-sitosterol and ursolic acid [a pentacyclic terpenoid, (Fig. 1)   bind to the PXR and CAR nuclear receptors and decrease the mRNA expression levels of Cyp3a1, Cyp2d1, and Cyp2c6. This is consistent with the findings of Chang et al. (2017), who found that ursolic acid (pentacyclic triterpene) had an antagonistic effect against PXR and CAR activity which is considered an ortholog of PXR and CAR in rats (Jones et al., 2000), resulting in a decrease of both mRNA and protein expressions of CYP3A4 and CYP2B6 (Chang et al., 2017;Picking et al., 2018). According to the previous report, downregulation of Cyp3a1, Cyp2d1, and Cyp2c6 mRNA correlated with decreasing in Cyp3a1, Cyp2d1, and Cyp2c6 activity (Zhou et al., 2019), suggesting that decreasing in Cyp3a1, Cyp2d1, and Cyp2c6 mRNA might cause drug-induced toxicity by the accumulation of drugs that are metabolized by Cyp3a1, Cyp2d1, and Cyp2c6 enzymes. The antagonistic effects of P. foetida and pentacyclic triterpenes (e.g., beta-sitosterol, lupeol, epifriedelinol, and ursolic acid) on PXR and CAR need to be further elucidated. Moreover, rat Cyp3a1, Cyp2d1, and Cyp2c6 isozymes correspond to the human CYP3A4, CYP2D6, and CYP2C9, respectively, and they are known to be involved in the most important phase I metabolism of most drugs. Therefore, the antagonistic effect of P. foetida on phase I metabolizing enzymes may increase the efficacy of anticancer drugs by reducing drug clearance through the downregulation of PXR and CAR (Xing et al., 2020). However, high intake of P. foetida might harm human health and need to be determined further.

CONCLUSION
Our study revealed that PFE has no subchronic toxic effects on the kidneys and liver in an animal model. However, long-term intake of P. foetida needs caution as it could lead to herb-drug interactions since P. foetida exhibits downregulation of mRNA expression levels and may alter the functions of the key CYP isozymes found in the rat's liver, including Cyp3a1, Cyp2c6, and Cyp2d1. This could lead to a reduction in CYP activity and may result in the accumulation of xenobiotics in the body. Nevertheless, several important aspects, such as the actual concentration of ursolic acid in P. foetida, coadministration of herb and drug, mechanism-based enzyme inhibition, and underlying mechanisms concerning upstream nuclear receptor and downstream protein levels, need to be further elucidated for a better understanding of the cytochrome P450 expression and the complete safety profile of P. foetida.

CONFLICT OF INTEREST
The authors report no financial or other conflicts of interest in this work.