Effects of Iron-tannic Acid Nanoparticles on Toxicity and Hepatocarcinogenicity in Rats

Abstract

Liver cancer is one of the most frequently occurring forms of cancers worldwide. Consequently, it has been associated with high global mortality rates. Early diagnosis and effective therapy remain a major challenge in diagnosis and treatment of liver cancer patients. Cancer nanotheranostics has become a promising strategy in recent years and metal-polyphenol nanoparticles appear to represent a good candidate for nanotheranostic agents. It has been reported that iron-tannic acid nanoparticles (Fe-TA NPs) can provide MRI contrast enhancement in the hepatocellular carcinoma cell line and preneoplastic lesions in rat livers. Moreover, Fe-TA NPs have exhibited an anti-proliferative effect via autophagic cell death in the hepatocellular carcinoma cell line. The simple preparation and beneficial physicochemical properties of Fe-TA NPs are favorable for potential theranostic applications; however, a toxicity profile of Fe-TA NPs would still need to be assessed. reserv Acute and subacute toxicity tests were carried out to evaluate the systemic toxicity of Fe-TA NPs. Based on the maximum solubility of Fe-TA NPs, a dose of 55 mg/kg body weight (BW) was used in acute toxicity tests and administered through intravenous and intraperitoneal routes to rats. The LD2o value of the intravenous route was found to be 55 mg/kg BW, while the LDso value of both the intravenous and intraperitoneal routes was greater than 55 mg/kg BW. Accordingly, intraperitoneal administration of Fe-TA NPs may be safer than intravenous administration. In subacute toxicity test, Fe-TA NPs ranging from 0.22 - 5.5 mg/kg BW was administered intraperitoneally every 3 days for a total of 10 times. The effective duration of Fe-TA NPs was determined by their clearance from the rats' livers. Rats were sacrificed 24 hours after the last injection, whereas rats in the satellite groups were sacrificed after 14 days of observation. Repeated administration of Fe-TA NPs did not alter the body and organ weights of the rats, apart from producing a significant increase in the weights of seminal vesicles at a dose of 1.1 mg/kg BW. Since the growth and activity of these organs are under the control of sex hormones, this outcome may have been due to the hormonal action of Fe-TA NPs. Although neutrophil percentage was increased at doses of 1.1 and 5.5 mg/kg BW, monocyte percentages were decreased at doses of 0.22, 1.1 and 5.5 mg/kg BW. Importantly, these values were within the reference range. This may be due to the response to foreign particles which was confirmed in previous studies focusing on the presence of increased numbers of Kupffer cells in rat livers following NPs treatment. Other hematological parameters were also determined to be within the reference range. Moreover, serum biochemical parameters were found within the reference range with the exception of serum potassium levels, which indicated significant increases at a dose of 5.5 mg/kg BW. However, the levels returned to within the reference range in the satellite groups. This would likely be due to the interaction of Fe-TA NPs with ion channels. Serum iron parameters and liver ferritin content were not changed, which may have been due to low concentrations of iron and its rapid clearance from circulation. The hepatocarcinogenicity of Fe-TA NPs in rats was assessed through medium- term carcinogenicity tests. Glutathione S-transferase placental form (GST-P) positive foci were used as endpoint preneoplastic markers. Rats received Fe-TA NPs ranging from 0.55 - 17.5 mg/kg BW intraperitoneally once a week for 10 weeks after undergoing a partial hepatectomy to induce regenerative proliferation and increase the sensitivity of the tests. Fe-TA NPs did not induce hepatic GST-P positive foci formation in rats, indicating a lack of hepatocarcinogenicity as well as genotoxicity. In addition, Fe-TA NPs did not alter the number of apoptotic cells and PCNA-positive cells in rat livers, which serve as a cell proliferating marker. Fe-TA NPs administration did not alter serum and liver iron parameters in rats that were evaluated in samples collected at 24 hours after the last injections. As oxidative stress plays a critical role in carcinogenesis, the effects of Fe-TA NPs on the activities of antioxidant enzymes and glutathione levels in rat livers were further investigated. Fe-TA NPs at a dose of 0.55 mg/kg BW decreased catalase (CAT) activity, whereas a dose of 17.5 mg/kg BW could induce antioxidant response as indicated by increased total glutathione content and CAT activity in rat livers. The activity of CAT, which was recovered when concentrations of Fe-TA NPs were increased, may have been due to the alleviation of an oxidative stress response. Carcinogenesis involves initiation, promotion, and progression stages. Although Fe-TA NPs did not play a role as tumor initiators, their non-genotoxic action needs to be considered. Therefore, diethylnitrosamine (DEN) was designated as an initiator in studies of the promoting effects of Fe-TA NPs on DEN-initiated hepatocarcinogenesis in rats. After administering triple intraperitoneal injections of DEN and partial hepatectomy, Fe- TA NPs injections were then administered every week for a total of 10 times. DEN decreased serum iron and transferrin saturation but did not alter liver iron content and ferritin levels in rats. Fe-TA NPs at doses of 1.75 and 17.5 mg/kg BW decreased total iron binding capacity but increased the liver iron content. However, these dosages did not affect liver ferritin levels in DEN-initiated rats. Notably, the treatment of Fe-TA NPs at 1.75 mg/kg BW enhanced both the number and area of GST-P positive foci in the livers of DEN-initiated rats indicating a promoting effect on hepatocarcinogenesis. Furthermore, the number of apoptotic cells was suppressed but PCNA-positive cells were increased favoring the net growth of GST-P positive foci. It can be suggested that Fe-TA NPs at 1.75 mg/kg BW promoted DEN-induced hepatocarcinogenesis in rats through the disruption of a balance of cell apoptosis and proliferation. Since Fe-TA NPs did not result in genotoxicity, the promotion mechanism may likely have occurred through nongenotoxic mechanisms. The alteration of gene expression and the changes in protein function caused by oxidative modification affect cell survival and proliferation. Hence, antioxidant parameters in DEN-initiated rat livers were evaluated. DEN injections increased the activity of CAT in the livers but did not modulate the activities of glutathione reductase (GR), glutathione peroxidase (GPx) and the oxidized to reduced glutathione ratios in rat livers. CAT activity was increased by 17.5 mg/kg BW of Fe-TA NPs; however, GPx activity was decreased at different doses of Fe-TA NPs in DEN-initiated rats. It can be suggested that GPx was inactivated in the presence of reactive nitrogen species. In addition, GR activity was not altered by Fe-TA NPs treatment in DEN-initiated rats. Furthermore, Fe-TA NPs treatment decreased the ratio of oxidized glutathione and reduced glutathione content indicating antioxidant response by de novo synthesis. Thus, it was assumed that high levels of oxidative stress at 1.75 mg/kg BW can be related to increased cell proliferation. Since Fe-TA NPs can be degraded in the presence of H2O2 through the disruption of coordinated interactions between iron and TA, it can be proposed that Fe-TA NPs would be dissociated into ferric iron and TA levels in the oxidative environment of DEN- treated livers. TA could reduce ferric iron to ferrous iron, which would then cause a Fenton reaction favoring oxidative stress and a greater dissociation of Fe-TA NPs. These oxidative conditions could enhance cell survival and proliferation leading to increased formation of preneoplastic lesions. In conclusion, Fe-TA NPs presented no obvious systemic toxicity in rats. The LD20 value of the intravenous route was 55 mg/kg BW, while the LDso value of the intravenous and intraperitoneal routes was greater than 55 mg/kg BW. This would indicate that the intraperitoneal administration was a safer route for the administration of Fe-TA NPs in rat models. Moreover, repeated doses of Fe-TA NPs were not determined to be toxic to various vital organs and did not induce preneoplastic lesions in rat livers. Fe-TA NPs also modulated hepatic antioxidant systems in rats depending upon the underlying cellular regulatory mechanisms. However, Fe-TA NPs at a certain dose could promote the formation of preneoplastic lesions in the livers of diethynitrosamine-initiated rats by the suppression of apoptosis and the enhancement of cell proliferation. Therefore, Fe-TA NPs may be safe for diagnostic applications as an MRI contrast agent, whereas therapeutic applications would require the frequent and repeated administration of Fe-TA NPs and further studies on the effects of Fe-TA NPs on immune cells, androgen levels and potassium homeostasis, as well as those incorporating dosage adjustments and species variations in relation to non-genotoxic effects, are needed to evaluate the overall safety of this recommendation. Moreover, the study of the effect of Fe-TA NPs on the advanced stages of hepatocarcinogenesis would be of considerable value.