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Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat
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Experimental and Toxicologic Pathology xxx (2015) xxx–xxx
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Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat Xue-yan Pu, Jia-ying Shen, Zhong-ping Deng, Ze-an Zhang* Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2016 Received in revised form 26 February 2016 Accepted 21 March 2016
Many Aristolochia species herbal drugs, used for diseases treatment since antiquity, contain active component aristolochic acid mixture, which consists of aristolochic acid I and II. However, it remains unclear whether aristolochic acid I is gastrotoxic, though evidence has shown that aristolochic acid mixture is nephrotoxic, carcinogenic, and genotoxic. The present study aimed to investigate the gastrotoxicity in rats treated with aristolochic acid I alone. Four groups of rats were orally administrated with vehicle (1% NaHCO3), or 30 mg, 60 mg, and 90 mg/kg/day of aristolochic acid I for twelve days. The results showed that aristolochic acid I can induce obvious body weight loss, forestomach injury characterized by necrosis, ulcer, hyperkeratosis, and hyperplasia of epithelial cells. The severity of these forestomach lesions was presented in a dose-dependent mode. Meanwhile, only non-specific, slight renal tubule degeneration, and occasionally single necrotic epithelial cell were found in aristolochic acid Itreated rats’ kidney. These resulst indicated aristolochic acid I had obvious gastrotoxicity, and such aristolochic acid I-induced forestomach toxicity probably presented much prior to kidney injury. Such irritation lesions may play a partial role in gastric cancer development of rats induced by aristolochic acid. Therefore, these results expanded our understanding on the digestive system toxicity of aristolochic acid I. ã 2016 Elsevier GmbH. All rights reserved.
Keywords: Aristolochic acid I Stomach Kidney Histology Toxicity Rat
1. Introduction Aristolochic acids (AAs) are natural products derived from taxa in the Aristolochiaceae. They originate primarily from the genera Aristolochia and Asarum (NTP, 2008). Traditional Chinese Medicine containing aristolochic acid (AA) has been used extensively in hepatitis, uropoietic system and cardiovascular system (Zhang et al., 2013). Epidemiological studies showed that AA exposure is associated with a high risk of nephrotoxicity and upper urinary tract carcinoma (UUC) (De Broe, 1999; Grollman et al., 2007; Nortier et al., 2000). This disease, named aristolochic acid nephropathy (AAN) was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi ((Michl et al.,
Abbreviations: AAs, aristolochic acids; AA, aristolochic acid; AA I, aristolochic acid I; AA II, aristolochic acid II; UUC, upper urinary tract carcinoma; AAN, aristolochic acid nephropathy; TP, total protein; ALB, albumin; BUN, blood urea nitrogen; CREA, creatinine; HE, hematoxylin and eosin. * Corresponding author at: Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Room 1403, No. 1 Building, Cai Lun Road, Zhangjiang, Pudong, Shanghai 201203, PR China. E-mail address: [email protected] (Z.-a. Zhang).
2014). Subsequently, new sporadic AAN cases were regularly reported throughout the world (Debelle et al., 2008). It has been estimated that 100 million people may be at risk of developing AAN in China alone (Hu et al., 2004). AA is a mixture containing structurally-related aristolochic acid I (AA I) and aristolochic acid II (AA II). Additionally, the content of AA I is highest among AA mixture, and it is the most representative substance in AA compound. Research showed that AA I induced tubular cell necrosis and interstitial fibrosis in the renal cortex, but AA II only resulted in minimal changes in the renal cortex of the male C3H/He mice (Shibutani et al., 2007). DNA adduct induced by AA I in stomach was significantly higher than AA II (Shibutani et al., 2007). These results indicated AA I was the main reason for kidney injury, and may play a critical role in gastric toxicity. Previous report showed that intragastric administration of 10 mg/kg/day AA (77.24% AA I and 22.18% AA II) in rats caused isolated swollen epithelial cells only twenty-four hours after the first administration, which showed vacuolation of the cytoplasm and cystic distension of the nucleus (Mengs, 1983). Branched papilloma of the forestomach was observed after 90 days (Mengs, 1983). Later, the same researcher reported that the rats was give once the sodium salt of AA mixture by gastric intubation at a dose of 200 mg/kg, marked hyperplasia with hyperkeratosis of the
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Please cite this article in press as: X.- Pu, et al., Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.03.003
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forestomach epithelium was developed after 14 days (Mengs, 1987). It is also reported that AA mixture causes DNA adducts formation (dA-AAI, dG-AAI, dA-AAII, dG-AAII) and is carcinogenic in rat forestomach and stomach (Stiborová et al., 1994; NTP, 2014). However, as AA mixture (AA-I and AA-II) was used in these studies, it was not possible to determine whether one or both components of AA contributed to the gastrotoxic effect. Although there are many researches about the AA nephrotoxicity, most ones focused on AA mixture. As for digestive system toxicity, there is no report to define the effect of individual AA I on stomach. For this purpose, the effect of AA I on rat stomach was investigated via histology in the present study, and the nephrotoxic effect of AA I was evaluated as well.
of Sciences), with body weight initially ranging from 220 to 230 g. All animals were used and cared in compliance with the local Institutional Animal Care and Use Committee (Centre for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine). Twelve Wistar rats were kept in plastic cages in SPF animal room(temperature: 20–25 C, relative humidity: 40– 70%). Standard food and drinking water were available ad libitum. Rats were randomly divided into 4 groups with 3 rats per group. Three groups of rats were given AA I by gastric intubation at dose of 30, 60 and 90 mg/kg/day for 12 days, respectively. Control rats were given an equivalent amount (10 ml/kg) of 1% NaHCO3 (control vehicle). 2.3. Clinical pathological data collection and detection
2. Materials and methods 2.1. Reagents AA I was supplied by Sigma Aldrich, and the purity is no less than 90%. Sodium bicarbonate (NaHCO3) was purchase from Sinopham Chemical Reagent Co., Ltd. Water was supplied by Millipore Elix, and sterilized to use. One percent of NaHCO3 solution (1% NaHCO3), as a control vehicle, was prepared in sterilized water, and AA I solution was prepared in 1% NaHCO3 solution. 2.2. Laboratory animals and treatment The laboratory animals used were 8–9 weeks old male healthy Wistar rats (Shanghai Laboratory Animal Centre, Chinese Academy
On day 1, 2, 4, 6, 8, 10 and 12, body weight was measured just before treatment, and the administration volume was adjusted according to the body weight. Blood was taken from tail vein, and 6 h urine was collected after each treatment on day 4, 8 and 12. All blood and urine were proceeded to detect levels of total protein (TP), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA), using automatic biochemical analyzer (HITACHI 7080). 2.4. Histology All rats were euthanized on day 12. Blood was collected from abdominal aorta. Kidney and stomach of each rat were removed and fixed in 10% neutral buffered formalin for at least 24 h, then dehydrated, embedded, sectioned with 4 mm in thickness, and stained with hematoxylin and eosin (HE) for histological analysis.
Fig. 1. Forestomach lesions induced by AA I. (A) No histological changes were founded in the stomach of control rats. (B) Focal ulceration, and marked hyperplasia with hyperkeratosis of forestomach epithelium were showed (30 mg/kg AA I). (C) Ulceration was obvious, with hyperplasia, and hyperkeratosis of forestomach epithelium (60 mg/ kg AA I). (D) Massive ulceration, and marked hyperplasia with hyperkeratosis of the forestomach epithelium were presented (90 mg/kg AA I). H&E, 50.
Please cite this article in press as: X.- Pu, et al., Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.03.003
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Fig. 2. The effect of AA I on renal histology. (A) No histological lesions were observed in control rats. Minimal degeneration in renal tubules with a few single cell necrosis in epithelium was shown in 30 mg/kg (B), 60 mg/kg (C), and 90 mg/kg (D) AA I-treated rats. H&E, 200 (left panel), 400 (right panel).
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increased number of epithelial cells, and resulted in an increased thickness of forestomach mucosa. In 30 mg/kg dose level, focal ulceration, marked hyperplasia with hyperkeratosis of the forestomach epithelium was observed after 12 days (Fig. 1B). In 60 mg/kg dose level, the hyperplasia with hyperkeratosis changes were still at a marked degree, ulceration is obvious (Fig. 1C). In 90 mg/kg dose level, massive ulceration, marked hyperplasia with hyperkeratosis of the forestomach epithelium was observed (Fig. 1D). The severity of these lesions was showed in a dose-dependent mode. In control rats, no histopathological changes were found in stomach, the mucosa is good, and the cell layer is very clear (Fig. 1A). 3.2. Renal histology
Fig. 3. The changes of body weight in AA I-treated rats. Body weight decreased from day 2 to day 12. Compared to control rats, body weights in all AA I-treated rats were significantly decreased from day 8 to day 12. Body weights of rats treated by 60 mg/ kg AA I were obviously declined on day 6. *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs vehicle.
2.5. Statistical analysis All data were expressed as mean SD values, and analyzed by Student’s t-test (t-test). p < 0.05 is considered as significant changes. 3. Results 3.1. Gastric histopathology Forestomach and glandular stomach were assessed via histology. Lesions was observed in forestomach, while there is no histological changes found in glandular stomach. As shown in Fig. 1, there was marked hyperplasia with hyperkeratosis, mild to marked ulceration in forestomach epithelium of rats treated by 30, 60 and 90 mg/kg AA I. The histological appearance of ulceration in forestomach was a mixture of necrosis with acute and chronic inflammation, slight edema in submucosa and fibrosis in base of ulcer. Hyperkeratosis showed a clear increase of the superficial non-nucleated keratin layer. Hyperplasia was characterized by an
Table 1 The effect of AA I on renal clinical pathology in serum. Time
Group
TP(g/L)
ALB(g/L)
BUN(mmol/L) CREA(umol/L)
D4
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
61.00 1.18 59.53 4.58 62.17 0.99 61.87 1.27
33.10 0.20 33.30 1.15 33.53 0.40 33.77 0.55
4.57 0.91 4.13 0.93 4.53 0.65 4.27 0.76
21.33 3.21 18.33 0.58 17.33 0.58 20.67 0.58
D8
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
63.80 1.80 55.30 2.60 55.20 2.60 57.80 2.80
33.90 0.30 41.60 5.30 30.30 1.70 30.40 1.10
5.30 0.60 3.70 0.30 4.10 0.70 8.30 7.70
22.30 2.10 18.00 0.00 17.30 2.10 48.70 54.10
D12
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
52.13 1.33 48.17 0.83 50.30 2.03 50.80 1.56
28.87 1.00 28.77 0.75 29.20 0.96 29.13 0.29
5.63 0.31 3.87 0.21 4.90 0.26 16.6 15.94
25.33 3.51 22.33 3.21 20.67 4.73 36.67 26.39
Note: D = day, TP = total protein; ALB = albumin; BUN = blood urea nitrogen; CREA = creatinine.
Only minimal degeneration with a few single cell necrosis was observed in kidneys of AA I-treated rats as shown in Fig. 2, and there was minimal renal tubule degeneration in one rat dosed at 30 mg/kg group(Fig. 2B), and all rats dosed at 60 mg/kg groups (Fig. 2C) and 90 mg/kg groups(Fig. 2D). There were no histological changes founded in kidneys of control rats (Fig. 2A). 3.3. Body weight and clinical pathology The effect of AA I on body weight were shown in Fig. 3. There was a decrease trend in body weight for all AA I-treated rats from day 2 to day 12. The body weighs of rats treated by 60 mg/kg on day 6 to day 12, 30 mg/kg and 90 mg/kg AA I on day 8 to day 12 were significantly lower than that of control rats (p < 0.05, p < 0.01, p < 0.001). As shown in Table 1, though the levels of serum ALB and TP had a decrease trend in rats administrated by 60 mg/kg and 90 mg/kg AA I, and the levels of CREA showed an increase in rats treated by 90 mg/kg AA I on day 8 and day 12, they were no statistical difference compared to those in control rats. Furthermore, the levels of BUN in serum and urine, ALB, TP and CREA in urine were no obvious changes between AA I-treated rats and control rats (Tables 1 and 2). 4. Discussion Contrast to a large pool of evidence about AA mixture-induced nephrotoxicity (Mengs, 1987; Mengs and Stotzem, 1993; Mei et al., 2006; Sidorenko et al., 2012), there is no report on the impact of individual AA I on stomach. In the present study, our results showed that acute exposure to AA I via oral gavage can obviously induce gastric lesions in rats, characterized by ulceration, hyperplasia, and hyperkeratosis of forestomach epithelium, and inflammatory cells infiltration in a dose-dependent mode. Previous reports showed when the rats were given mixture of AA I and AA II by oral gavage, massive necrosis of the forestomach epithelium with edema and leukocytes infiltration after 48 h, and hyperplasia with hyperkeratosis of the forestomach epithelium after day 14 were presented (Mengs, 1983, 1987). Compared to the presence of extensive gastric necrosis induced by 10 mg/kg AA mixture just on day 2 (Mengs, 1983), it is on day 12 that focal (30 mg/kg AA I) and extensive/severe (60 and 90 mg/kg AA I) gastric injury was founded in the present study. Combined with our observations, these results indicated that AA I has obvious gastrotoxicity, and such toxic effect exerted by AA mixture is more powerful than individual AA I. Interestingly, while massive necrosis was observed on day 2, hyperplasia and hyperkeratosis were not presented until on day 14 in forestomach of rats treated by 10 mg/kg AA mixture (Mengs, 1983), our observations showed there were only sporadic necrotic cells, but obvious hyperplasia and hyperkeratosis founded on day 12 in forestomach of rats treated by 30 mg/kg AA I. This suggested
Please cite this article in press as: X.- Pu, et al., Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.03.003
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Table 2 The effect of AA I on renal clinical pathology in urine. Time
Group
TP(g/L)
ALB(g/L)
BUN(mmol/L)
CREA(umol/L)
D4
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
9.97 1.10 9.50 3.39 5.77 0.47 7.10 1.050
0.23 0.06 0.37 0.23 0.13 0.06 0.30 0.00
479.43 54.09 470.05 257.17 413.83 49.80 552.83 199.53
3389.67 568.99 4744.67 2435.92 3345.67 974.31 4759.33 1392.42
D8
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
4.27 0.15 4.43 0.42 5.23 0.15 5.33 0.68
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
310.67 41.82 213.40 28.60 417.60 80.75 215.77 28.85
2201.33 552.87 1453.00 219.50 3096.00 1114.40 2101.33 468.89
D12
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
4.57 0.72 4.67 0.21 4.50 0.36 6.70 2.30
0.10 0.17 0.03 0.06 0.00 0.00 0.27 0.23
361.57 188.03 180.90 29.88 169.60 88.22 341.53 348.19
2892.67 1487.14 1517.33 408.30 1437.00 597.64 2131.67 1968.76
Note: D = day, TP = total protein; ALB = albumin; BUN = blood urea nitrogen; CREA = creatinine.
that AA I possibly be the main contributor for inducing hyperplasia and hyperkeratosis of forestomach. Previous report showed that among AA mixture, AA I was mainly responsible for nephrotoxicity (Shibutani et al., 2007). In the present study, there was no obviously specific presence of kidney injury, characterized by only sporadic single cell necrosis of renal tubule epithelium, and by no obvious changes in clinical pathology (TP, ALB, BUN and CREA). This discrepancy may be resulted from different laboratory animal strain, and experimental procedures adopted in these two researches. But most probably, in our experimental conditions, the presence of nephrotoxic injury lagged behind the onset of gastric toxicity. On the other words, AA I induced the stomach injury earlier or faster than the development of AA I-induced kidney injury, which led no specific nephrotoxic manifestations to be observed. Thus the results indicated it was not kidney dysfunction but gastric injury that resulted in the loss of body weight in our research. The detailed mechanism/reason for body weight loss remains to be further elucidated. In previous reports, two different administration ways were utilized to establish AA-induced nephrotoxic model, including oral gavage or injection of peritoneal cavity. One report showed that a single high dose of 200 mg/kg AA mixture (77.24% AAI and 21.18% AAII) by oral gavage can induce renal injury with severe renal tubule necrosis in rats after 6 days (Mengs, 1987), while another research reported that a daily low dose of 10 mg/kg AA (40% AAI and 60% AAII) by peritoneal cavity injection for 5 days was capable of inducing kidney injury with swelling, brush border detachment and necrosis of a few epithelia (Lebeau et al., 2005). These results revealed that kidney injury caused by AA was developed faster or more severe by peritoneal cavity injection than by oral gavage. In our study, daily treatment of up to 90 mg/kg AA I by oral gavage can only induce non-specific and slight renal injury in rats, manifested by no obvious changes of renal clinical pathology parameters (TP, ALB, BUN and CREA), and occasional single necrosis and slight degeneration in the renal tubular epithelium. The absorption and metabolism process of AA I in digestive tract may be partly responsible for this absence or delay of specific and obvious renal injury. In liver AA mixture is metabolized to corresponding aristolactam I and aristolactam II under anaerobic conditions, while under aerobic conditions only metabolite is the Odemethylated derivative aristolochic acid Ia (AAIa) biotransformed from AA I (Schmeiser et al., 1986). Additionally, the hepatic cytochrome P450s play an important role in metabolizing aristolochic acid I to less toxic metabolites and thus have detoxification role in aristolochic adic I-induced kidney injury (Xiao et al., 2008). Therefore, the metabolism process and detoxification of AA I in liver may also contribute to the delay of
specific renal injury in the present study. This further suggested that the exposure routine of AA I or AA have an important effect on the presence or/and degree of renal injury. Combined with our observations of gastrotoxicity effect, it was showed that oral exposure was not the most desirable administration routine for establishing AA I or AA-induced nephrotoxic model in research. Aristolochic acids are metabolized to aristolactams, which are further metabolized to a cyclic N-acylnitrenium ion, a reactive intermediate that forms adducts with purine bases (adenine and guanine) in DNA (dA-AAI, dG-AAI, dA-AAII, and dG-AAII). DNA adducts have been detected in vivo in experimental animals and human (NTP, 2008, 2014). In animals, adducts have been detected in the forestomach and stomach, urinary tract (kidney and urinary bladder), liver, intestine, spleen, and lung. In humans, adducts have been detected in the urinary tract (kidney, ureter, and urinary bladder), liver, and non-target tissues such as pancreas, breast, and lung (NTP, 2008, 2014). The predominant adduct, dA-AAI, appears to be responsible for most of the mutagenic and carcinogenic properties of aristolochic acids (NTP, 2014). Taking our results into account, the stomach carcinogenic potential of AA mixture in rodents might be due to the combinational effect of DNA-adducts and irritation of stomach. 5. Conclusions Our results showed oral exposure to AA I can induce marked forestomach toxicity with non-specific renal toxicity in rats, and such gastric injury was presented earlier than specific renal injury. Our results also revealed that AA I-induced irritation lesions and DNA adducts in stomach may play a combinational role in gastric cancer development of rodents. Oral treatment, to some extent, was not the optimal way to establish nephrotoxicity model at least under our experimental conditions. Furthermore, it should be careful when the data from the orally-treated AA I or AA nephrotoxicity model is interpreted, because AA I-induced gastric injury may interfere with the readouts produced from the nephrotoxicity model. These results are helpful to understand the digestive system toxicity of AA I, and suggest most attention should be paid to the potential effect of AA I on explaining the readouts in AA mixture-induced nephrotoxicity model research. Conflict of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Please cite this article in press as: X.- Pu, et al., Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat, Exp Toxicol Pathol (2016), http://dx.doi.org/10.1016/j.etp.2016.03.003
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Acknowledgment This research was supported by Chinese National Science and Technology Major Project (Grant No. 2012ZX095001-002). References De Broe ME. On a nephrotoxic and carcinogenic slimming regimen. Am. J. Kidney. Dis. 1999;33(6):1171–3. Debelle FD, Vanherweghem JL, Nortier J.L.Frederic DD, Jean-Louis V, Joelle LN. Aristolochic acid nephropathy: a worldwide problem. Kidney Int. 2008;74 (2):158–69. Grollman AP, Shibutani S, Moriya M, Miller F, Wu L, Moll U, Suzuki N, Fernandes A, Rosenquist T, Medverec Z, Jakovina K, Brdar B, Slade N, Turesky RJ, Goodenough AK, Rieger R, Vukeli c M, Jelakovi c B. Aristolochic acid and the etiology of endemic (Balkan) nephropathy. Proc. Natl. Acad. Sci. U. S. A. 2007;104 (29):12129–34. Hu SL, Zhang HQ, Chan K, Mei QX. Studies on the toxicity of aristolochia manshuriensis (Guanmuton). Toxicology 2004;198(1–3):195–201. Lebeau C, Debelle FD, Arlt VM, Pozdzik A, De Prez EG, Phillips DH, DeschodtLanckman MM, Vanherweghem JL, Nortier J.L.Catherine L, Frédéric DD, Volker MA, Agnieszka P, Eric GD, David HP, Monique MD, Jean-Louis V, Joëlle LN. Early proximal tubule injure in experimental aristolochic acid nephropathy: functional and histological studies. Nephrol. Dial. Transplant. 2005;20 (11):2321–32. Mei N, Arlt VM, Phillips DH, Heflich RH, Chen T. DNA adduct formation and mutation induction by aristolochic acid in rat kidney and liver. Mutat. Res. 2006;602 (December (1–2)):83–91. Mengs U, Stotzem CD. Renal toxicity of aristolochic acid in rats as an example of nephrotoxicity testing in routine toxicology. Arch. Toxicol. 1993;67(5):307–11. Mengs U. On the histopathogenesis of rat forestomach carcinoma caused by aristolochic acid. Arch. Toxicol. 1983;52(3):209–20.
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Oral exposure to aristolochic acid I induces gastric histological lesions with non-specific renal injury in rat Xue-yan Pu, Jia-ying Shen, Zhong-ping Deng, Ze-an Zhang* Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 January 2016 Received in revised form 26 February 2016 Accepted 21 March 2016
Many Aristolochia species herbal drugs, used for diseases treatment since antiquity, contain active component aristolochic acid mixture, which consists of aristolochic acid I and II. However, it remains unclear whether aristolochic acid I is gastrotoxic, though evidence has shown that aristolochic acid mixture is nephrotoxic, carcinogenic, and genotoxic. The present study aimed to investigate the gastrotoxicity in rats treated with aristolochic acid I alone. Four groups of rats were orally administrated with vehicle (1% NaHCO3), or 30 mg, 60 mg, and 90 mg/kg/day of aristolochic acid I for twelve days. The results showed that aristolochic acid I can induce obvious body weight loss, forestomach injury characterized by necrosis, ulcer, hyperkeratosis, and hyperplasia of epithelial cells. The severity of these forestomach lesions was presented in a dose-dependent mode. Meanwhile, only non-specific, slight renal tubule degeneration, and occasionally single necrotic epithelial cell were found in aristolochic acid Itreated rats’ kidney. These resulst indicated aristolochic acid I had obvious gastrotoxicity, and such aristolochic acid I-induced forestomach toxicity probably presented much prior to kidney injury. Such irritation lesions may play a partial role in gastric cancer development of rats induced by aristolochic acid. Therefore, these results expanded our understanding on the digestive system toxicity of aristolochic acid I. ã 2016 Elsevier GmbH. All rights reserved.
Keywords: Aristolochic acid I Stomach Kidney Histology Toxicity Rat
1. Introduction Aristolochic acids (AAs) are natural products derived from taxa in the Aristolochiaceae. They originate primarily from the genera Aristolochia and Asarum (NTP, 2008). Traditional Chinese Medicine containing aristolochic acid (AA) has been used extensively in hepatitis, uropoietic system and cardiovascular system (Zhang et al., 2013). Epidemiological studies showed that AA exposure is associated with a high risk of nephrotoxicity and upper urinary tract carcinoma (UUC) (De Broe, 1999; Grollman et al., 2007; Nortier et al., 2000). This disease, named aristolochic acid nephropathy (AAN) was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi ((Michl et al.,
Abbreviations: AAs, aristolochic acids; AA, aristolochic acid; AA I, aristolochic acid I; AA II, aristolochic acid II; UUC, upper urinary tract carcinoma; AAN, aristolochic acid nephropathy; TP, total protein; ALB, albumin; BUN, blood urea nitrogen; CREA, creatinine; HE, hematoxylin and eosin. * Corresponding author at: Center for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine, Room 1403, No. 1 Building, Cai Lun Road, Zhangjiang, Pudong, Shanghai 201203, PR China. E-mail address: [email protected] (Z.-a. Zhang).
2014). Subsequently, new sporadic AAN cases were regularly reported throughout the world (Debelle et al., 2008). It has been estimated that 100 million people may be at risk of developing AAN in China alone (Hu et al., 2004). AA is a mixture containing structurally-related aristolochic acid I (AA I) and aristolochic acid II (AA II). Additionally, the content of AA I is highest among AA mixture, and it is the most representative substance in AA compound. Research showed that AA I induced tubular cell necrosis and interstitial fibrosis in the renal cortex, but AA II only resulted in minimal changes in the renal cortex of the male C3H/He mice (Shibutani et al., 2007). DNA adduct induced by AA I in stomach was significantly higher than AA II (Shibutani et al., 2007). These results indicated AA I was the main reason for kidney injury, and may play a critical role in gastric toxicity. Previous report showed that intragastric administration of 10 mg/kg/day AA (77.24% AA I and 22.18% AA II) in rats caused isolated swollen epithelial cells only twenty-four hours after the first administration, which showed vacuolation of the cytoplasm and cystic distension of the nucleus (Mengs, 1983). Branched papilloma of the forestomach was observed after 90 days (Mengs, 1983). Later, the same researcher reported that the rats was give once the sodium salt of AA mixture by gastric intubation at a dose of 200 mg/kg, marked hyperplasia with hyperkeratosis of the
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forestomach epithelium was developed after 14 days (Mengs, 1987). It is also reported that AA mixture causes DNA adducts formation (dA-AAI, dG-AAI, dA-AAII, dG-AAII) and is carcinogenic in rat forestomach and stomach (Stiborová et al., 1994; NTP, 2014). However, as AA mixture (AA-I and AA-II) was used in these studies, it was not possible to determine whether one or both components of AA contributed to the gastrotoxic effect. Although there are many researches about the AA nephrotoxicity, most ones focused on AA mixture. As for digestive system toxicity, there is no report to define the effect of individual AA I on stomach. For this purpose, the effect of AA I on rat stomach was investigated via histology in the present study, and the nephrotoxic effect of AA I was evaluated as well.
of Sciences), with body weight initially ranging from 220 to 230 g. All animals were used and cared in compliance with the local Institutional Animal Care and Use Committee (Centre for Drug Safety Evaluation and Research, Shanghai University of Traditional Chinese Medicine). Twelve Wistar rats were kept in plastic cages in SPF animal room(temperature: 20–25 C, relative humidity: 40– 70%). Standard food and drinking water were available ad libitum. Rats were randomly divided into 4 groups with 3 rats per group. Three groups of rats were given AA I by gastric intubation at dose of 30, 60 and 90 mg/kg/day for 12 days, respectively. Control rats were given an equivalent amount (10 ml/kg) of 1% NaHCO3 (control vehicle). 2.3. Clinical pathological data collection and detection
2. Materials and methods 2.1. Reagents AA I was supplied by Sigma Aldrich, and the purity is no less than 90%. Sodium bicarbonate (NaHCO3) was purchase from Sinopham Chemical Reagent Co., Ltd. Water was supplied by Millipore Elix, and sterilized to use. One percent of NaHCO3 solution (1% NaHCO3), as a control vehicle, was prepared in sterilized water, and AA I solution was prepared in 1% NaHCO3 solution. 2.2. Laboratory animals and treatment The laboratory animals used were 8–9 weeks old male healthy Wistar rats (Shanghai Laboratory Animal Centre, Chinese Academy
On day 1, 2, 4, 6, 8, 10 and 12, body weight was measured just before treatment, and the administration volume was adjusted according to the body weight. Blood was taken from tail vein, and 6 h urine was collected after each treatment on day 4, 8 and 12. All blood and urine were proceeded to detect levels of total protein (TP), albumin (ALB), blood urea nitrogen (BUN), and creatinine (CREA), using automatic biochemical analyzer (HITACHI 7080). 2.4. Histology All rats were euthanized on day 12. Blood was collected from abdominal aorta. Kidney and stomach of each rat were removed and fixed in 10% neutral buffered formalin for at least 24 h, then dehydrated, embedded, sectioned with 4 mm in thickness, and stained with hematoxylin and eosin (HE) for histological analysis.
Fig. 1. Forestomach lesions induced by AA I. (A) No histological changes were founded in the stomach of control rats. (B) Focal ulceration, and marked hyperplasia with hyperkeratosis of forestomach epithelium were showed (30 mg/kg AA I). (C) Ulceration was obvious, with hyperplasia, and hyperkeratosis of forestomach epithelium (60 mg/ kg AA I). (D) Massive ulceration, and marked hyperplasia with hyperkeratosis of the forestomach epithelium were presented (90 mg/kg AA I). H&E, 50.
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Fig. 2. The effect of AA I on renal histology. (A) No histological lesions were observed in control rats. Minimal degeneration in renal tubules with a few single cell necrosis in epithelium was shown in 30 mg/kg (B), 60 mg/kg (C), and 90 mg/kg (D) AA I-treated rats. H&E, 200 (left panel), 400 (right panel).
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increased number of epithelial cells, and resulted in an increased thickness of forestomach mucosa. In 30 mg/kg dose level, focal ulceration, marked hyperplasia with hyperkeratosis of the forestomach epithelium was observed after 12 days (Fig. 1B). In 60 mg/kg dose level, the hyperplasia with hyperkeratosis changes were still at a marked degree, ulceration is obvious (Fig. 1C). In 90 mg/kg dose level, massive ulceration, marked hyperplasia with hyperkeratosis of the forestomach epithelium was observed (Fig. 1D). The severity of these lesions was showed in a dose-dependent mode. In control rats, no histopathological changes were found in stomach, the mucosa is good, and the cell layer is very clear (Fig. 1A). 3.2. Renal histology
Fig. 3. The changes of body weight in AA I-treated rats. Body weight decreased from day 2 to day 12. Compared to control rats, body weights in all AA I-treated rats were significantly decreased from day 8 to day 12. Body weights of rats treated by 60 mg/ kg AA I were obviously declined on day 6. *: p < 0.05, **: p < 0.01, ***: p < 0.001 vs vehicle.
2.5. Statistical analysis All data were expressed as mean SD values, and analyzed by Student’s t-test (t-test). p < 0.05 is considered as significant changes. 3. Results 3.1. Gastric histopathology Forestomach and glandular stomach were assessed via histology. Lesions was observed in forestomach, while there is no histological changes found in glandular stomach. As shown in Fig. 1, there was marked hyperplasia with hyperkeratosis, mild to marked ulceration in forestomach epithelium of rats treated by 30, 60 and 90 mg/kg AA I. The histological appearance of ulceration in forestomach was a mixture of necrosis with acute and chronic inflammation, slight edema in submucosa and fibrosis in base of ulcer. Hyperkeratosis showed a clear increase of the superficial non-nucleated keratin layer. Hyperplasia was characterized by an
Table 1 The effect of AA I on renal clinical pathology in serum. Time
Group
TP(g/L)
ALB(g/L)
BUN(mmol/L) CREA(umol/L)
D4
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
61.00 1.18 59.53 4.58 62.17 0.99 61.87 1.27
33.10 0.20 33.30 1.15 33.53 0.40 33.77 0.55
4.57 0.91 4.13 0.93 4.53 0.65 4.27 0.76
21.33 3.21 18.33 0.58 17.33 0.58 20.67 0.58
D8
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
63.80 1.80 55.30 2.60 55.20 2.60 57.80 2.80
33.90 0.30 41.60 5.30 30.30 1.70 30.40 1.10
5.30 0.60 3.70 0.30 4.10 0.70 8.30 7.70
22.30 2.10 18.00 0.00 17.30 2.10 48.70 54.10
D12
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
52.13 1.33 48.17 0.83 50.30 2.03 50.80 1.56
28.87 1.00 28.77 0.75 29.20 0.96 29.13 0.29
5.63 0.31 3.87 0.21 4.90 0.26 16.6 15.94
25.33 3.51 22.33 3.21 20.67 4.73 36.67 26.39
Note: D = day, TP = total protein; ALB = albumin; BUN = blood urea nitrogen; CREA = creatinine.
Only minimal degeneration with a few single cell necrosis was observed in kidneys of AA I-treated rats as shown in Fig. 2, and there was minimal renal tubule degeneration in one rat dosed at 30 mg/kg group(Fig. 2B), and all rats dosed at 60 mg/kg groups (Fig. 2C) and 90 mg/kg groups(Fig. 2D). There were no histological changes founded in kidneys of control rats (Fig. 2A). 3.3. Body weight and clinical pathology The effect of AA I on body weight were shown in Fig. 3. There was a decrease trend in body weight for all AA I-treated rats from day 2 to day 12. The body weighs of rats treated by 60 mg/kg on day 6 to day 12, 30 mg/kg and 90 mg/kg AA I on day 8 to day 12 were significantly lower than that of control rats (p < 0.05, p < 0.01, p < 0.001). As shown in Table 1, though the levels of serum ALB and TP had a decrease trend in rats administrated by 60 mg/kg and 90 mg/kg AA I, and the levels of CREA showed an increase in rats treated by 90 mg/kg AA I on day 8 and day 12, they were no statistical difference compared to those in control rats. Furthermore, the levels of BUN in serum and urine, ALB, TP and CREA in urine were no obvious changes between AA I-treated rats and control rats (Tables 1 and 2). 4. Discussion Contrast to a large pool of evidence about AA mixture-induced nephrotoxicity (Mengs, 1987; Mengs and Stotzem, 1993; Mei et al., 2006; Sidorenko et al., 2012), there is no report on the impact of individual AA I on stomach. In the present study, our results showed that acute exposure to AA I via oral gavage can obviously induce gastric lesions in rats, characterized by ulceration, hyperplasia, and hyperkeratosis of forestomach epithelium, and inflammatory cells infiltration in a dose-dependent mode. Previous reports showed when the rats were given mixture of AA I and AA II by oral gavage, massive necrosis of the forestomach epithelium with edema and leukocytes infiltration after 48 h, and hyperplasia with hyperkeratosis of the forestomach epithelium after day 14 were presented (Mengs, 1983, 1987). Compared to the presence of extensive gastric necrosis induced by 10 mg/kg AA mixture just on day 2 (Mengs, 1983), it is on day 12 that focal (30 mg/kg AA I) and extensive/severe (60 and 90 mg/kg AA I) gastric injury was founded in the present study. Combined with our observations, these results indicated that AA I has obvious gastrotoxicity, and such toxic effect exerted by AA mixture is more powerful than individual AA I. Interestingly, while massive necrosis was observed on day 2, hyperplasia and hyperkeratosis were not presented until on day 14 in forestomach of rats treated by 10 mg/kg AA mixture (Mengs, 1983), our observations showed there were only sporadic necrotic cells, but obvious hyperplasia and hyperkeratosis founded on day 12 in forestomach of rats treated by 30 mg/kg AA I. This suggested
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Table 2 The effect of AA I on renal clinical pathology in urine. Time
Group
TP(g/L)
ALB(g/L)
BUN(mmol/L)
CREA(umol/L)
D4
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
9.97 1.10 9.50 3.39 5.77 0.47 7.10 1.050
0.23 0.06 0.37 0.23 0.13 0.06 0.30 0.00
479.43 54.09 470.05 257.17 413.83 49.80 552.83 199.53
3389.67 568.99 4744.67 2435.92 3345.67 974.31 4759.33 1392.42
D8
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
4.27 0.15 4.43 0.42 5.23 0.15 5.33 0.68
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
310.67 41.82 213.40 28.60 417.60 80.75 215.77 28.85
2201.33 552.87 1453.00 219.50 3096.00 1114.40 2101.33 468.89
D12
Vehicle 30 mg/kg 60 mg/kg 90 mg/kg
4.57 0.72 4.67 0.21 4.50 0.36 6.70 2.30
0.10 0.17 0.03 0.06 0.00 0.00 0.27 0.23
361.57 188.03 180.90 29.88 169.60 88.22 341.53 348.19
2892.67 1487.14 1517.33 408.30 1437.00 597.64 2131.67 1968.76
Note: D = day, TP = total protein; ALB = albumin; BUN = blood urea nitrogen; CREA = creatinine.
that AA I possibly be the main contributor for inducing hyperplasia and hyperkeratosis of forestomach. Previous report showed that among AA mixture, AA I was mainly responsible for nephrotoxicity (Shibutani et al., 2007). In the present study, there was no obviously specific presence of kidney injury, characterized by only sporadic single cell necrosis of renal tubule epithelium, and by no obvious changes in clinical pathology (TP, ALB, BUN and CREA). This discrepancy may be resulted from different laboratory animal strain, and experimental procedures adopted in these two researches. But most probably, in our experimental conditions, the presence of nephrotoxic injury lagged behind the onset of gastric toxicity. On the other words, AA I induced the stomach injury earlier or faster than the development of AA I-induced kidney injury, which led no specific nephrotoxic manifestations to be observed. Thus the results indicated it was not kidney dysfunction but gastric injury that resulted in the loss of body weight in our research. The detailed mechanism/reason for body weight loss remains to be further elucidated. In previous reports, two different administration ways were utilized to establish AA-induced nephrotoxic model, including oral gavage or injection of peritoneal cavity. One report showed that a single high dose of 200 mg/kg AA mixture (77.24% AAI and 21.18% AAII) by oral gavage can induce renal injury with severe renal tubule necrosis in rats after 6 days (Mengs, 1987), while another research reported that a daily low dose of 10 mg/kg AA (40% AAI and 60% AAII) by peritoneal cavity injection for 5 days was capable of inducing kidney injury with swelling, brush border detachment and necrosis of a few epithelia (Lebeau et al., 2005). These results revealed that kidney injury caused by AA was developed faster or more severe by peritoneal cavity injection than by oral gavage. In our study, daily treatment of up to 90 mg/kg AA I by oral gavage can only induce non-specific and slight renal injury in rats, manifested by no obvious changes of renal clinical pathology parameters (TP, ALB, BUN and CREA), and occasional single necrosis and slight degeneration in the renal tubular epithelium. The absorption and metabolism process of AA I in digestive tract may be partly responsible for this absence or delay of specific and obvious renal injury. In liver AA mixture is metabolized to corresponding aristolactam I and aristolactam II under anaerobic conditions, while under aerobic conditions only metabolite is the Odemethylated derivative aristolochic acid Ia (AAIa) biotransformed from AA I (Schmeiser et al., 1986). Additionally, the hepatic cytochrome P450s play an important role in metabolizing aristolochic acid I to less toxic metabolites and thus have detoxification role in aristolochic adic I-induced kidney injury (Xiao et al., 2008). Therefore, the metabolism process and detoxification of AA I in liver may also contribute to the delay of
specific renal injury in the present study. This further suggested that the exposure routine of AA I or AA have an important effect on the presence or/and degree of renal injury. Combined with our observations of gastrotoxicity effect, it was showed that oral exposure was not the most desirable administration routine for establishing AA I or AA-induced nephrotoxic model in research. Aristolochic acids are metabolized to aristolactams, which are further metabolized to a cyclic N-acylnitrenium ion, a reactive intermediate that forms adducts with purine bases (adenine and guanine) in DNA (dA-AAI, dG-AAI, dA-AAII, and dG-AAII). DNA adducts have been detected in vivo in experimental animals and human (NTP, 2008, 2014). In animals, adducts have been detected in the forestomach and stomach, urinary tract (kidney and urinary bladder), liver, intestine, spleen, and lung. In humans, adducts have been detected in the urinary tract (kidney, ureter, and urinary bladder), liver, and non-target tissues such as pancreas, breast, and lung (NTP, 2008, 2014). The predominant adduct, dA-AAI, appears to be responsible for most of the mutagenic and carcinogenic properties of aristolochic acids (NTP, 2014). Taking our results into account, the stomach carcinogenic potential of AA mixture in rodents might be due to the combinational effect of DNA-adducts and irritation of stomach. 5. Conclusions Our results showed oral exposure to AA I can induce marked forestomach toxicity with non-specific renal toxicity in rats, and such gastric injury was presented earlier than specific renal injury. Our results also revealed that AA I-induced irritation lesions and DNA adducts in stomach may play a combinational role in gastric cancer development of rodents. Oral treatment, to some extent, was not the optimal way to establish nephrotoxicity model at least under our experimental conditions. Furthermore, it should be careful when the data from the orally-treated AA I or AA nephrotoxicity model is interpreted, because AA I-induced gastric injury may interfere with the readouts produced from the nephrotoxicity model. These results are helpful to understand the digestive system toxicity of AA I, and suggest most attention should be paid to the potential effect of AA I on explaining the readouts in AA mixture-induced nephrotoxicity model research. Conflict of interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
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