The benefits of green tea polyphenols (GTP) consumption have been scientifically established. GTP are a source of valuable phytochemical compounds whose consumption may improve health and constitute a protection factor against some pathologies, especially cardiovascular disorders and cancer [1-2]. Green tea polyphenol catechins seem to be highly active molecules acting as prooxidant [3], antioxidant [2], anti-inflammatory [4] and anticancer [5] agents. Epigallocatechin gallate is the most widely studied compound [1]. Among the standardized preparations enriched in polyphenols is Polyphenon 60, which has been used in studies on inflammation-related colon carcinogenesis in mice [4] and in studies of catechin pharmacokinetics in healthy human beings [5]. The combination of epigallocatechin gallate and sulindac significantly enhanced apoptosis in rat colon, reducing the aberrant crypt foci [6]. Despite these positive effects, administration of concentrated green tea extracts has been shown to trigger certain adverse effects that rise concerns from a food safety point of view [7-9].

Plant ribosome-inactivating proteins (RIPs) are widespread throughout the plant kingdom [10,11] and are also present in fungi [12]. It is believed that they belong to the plant defence system against insects, viruses and fungi [13-15]. Interest in RIPs derives from their use in anticancer immunotoxins and conjugate preparations [16- 18]. Among these proteins are nigrin from elderberry (Sambucus nigra L.) [19] and ebulin from dwarf elder (Sambucus ebulus L.) [20]. Administering large amounts of nigrin b caused serious intestinal derangement that at high concentrations might lead to death [21]. In a previous light microscopy histological analysis, we reported that mice treated with sub-lethal doses of nigrin b revealed severe alteration of small intestine stem cells and transit-amplifying compartment (TAC) present in the crypts, displaying apoptotic-like morphology, including cell shrinkage and increased cytoplasmic eosinophilia. However, Paneth cells, which are located underneath TAC in the crypts, seemed to be spared [19,22]. A recent report from our laboratory used sub-lethal nigrin b administration to mice as a model for the study of vitamin B6 uptake [23]. The objective of the present research was to investigate whether administering GTP to nigrin binjured mice would affect the damaging action of sublethal nigrin b on the mouse intestinal mucosa, and the survival of the animals.

Oral administration of 16 or 30 mg/kg body weight of Polyphenon 60 to Swiss female mice had no visible effects (Figure 1). Likewise, intraperitoneal administration of 10 mg/kg nigrin did not affect animal survival or trigger apparent and visible damage (Figure 1). In contrast, oral administration of either 30 or 16 mg/kg Polyphenon 60 to mice treated simultaneously with 10 mg/kg body weight i.p. nigrin b reduced mice survival of animals sensitive to the treatment. Animals killed by the combined treatment showed dark areas in some segments of the small intestine probably by haemorrhage. It was noteworthy that the higher the Polyphenon 60 concentration in the combined treatment was, the shorter resulted to be the period to trigger lethal effects. At the highest concentration of Polyphenon 60 the combined treatment started to show lethal effects after 3 days, while at the lowest dose of Polyphenon 60, some animals started to die after 8 days. As we wanted to study only the early toxicity, the experiments were discontinued after 14 days once obtained a clear idea of the effect.

As shown in Figure 2, histological analysis revealed that mice treated with oral doses of 30 mg/kg Polyphenon 60 evidenced no intestinal derangement and, as in the control animals, showed well-formed Lieberkühn

crypts and villi (Figures 2(A) and (C)). In contrast, after 24 h of intraperitoneal administration of 10 mg/kg nigrin b, significant but reversible derangement of the small intestine was apparent, which led to the almost complete disappearance of the Lieberkühn crypts (Figure 2(D)). In this group, the epithelium of the crypts displayed a large number of dying cells showing apoptotic-like morphology including condensation of the chromatin with aggregation in peripheral masses, and ultimate formation of apoptotic bodies (Figure 2(D)); nevertheless the general structure of the intestinal villi was normal and their covering epithelium evidenced only minor shrinkage in the basal cytoplasm (Figure 2(D)). Simultaneous oral administration of 30 mg/kg Polyphenon 60 and intraperitoneal 10 mg/kg nigrin b revealed that the small intestines

were severely affected (Figure 2(B)), even more so than with nigrin b alone. Simultaneous administration of both agents caused almost complete destruction of the small intestine crypts. The structure of the large intestine was generally well preserved by that time (data not shown).

Numerous reports highlight the benefits of green tea consumption [26-28]. However, several detrimental effects associated to the ingestion of large amounts of green tea or preparations of green tea enriched with polyphenols have been reported [7,9]. Of particular concern is the hepatotoxicity presumably resulting from the ingestion of concentrated green tea extracts [8,29-31]. Concerning the mechanism of hepatotoxicity, it is not completely understood, but some components like catechins and their gallic acid esters, particularly epigallocatechin- 3-gallate which, under certain conditions such as fasting, can induce reactive oxygen species formation, and affect mitochondrial membrane potential [32], are likely to constitute an important part of such effect. A recent published review suggest than patterns of liver injury were hepatocellular in most cases, but cholestasis and a mixed pattern were also observed. Liver histology examination revealed inflammatory reactions, cholestasis, occasional steatosis and necrosis [33]. We therefore investigated the effects of simultaneously administering nigrin b (i.p.) and Polyphenon 60 (oral) on liver, lungs and kidney. While lungs and kidney were slightly congested (data not shown), the liver showed signs of haemorrhage (Figures 2(E) and (F)). Our results suggest that the apoptotic action of nigrin b on the small intestine crypts may be enhanced by the proapoptotic effects of polyphenols resulting in an increased rate of cell destruction. In fact, a synergistic apoptotic action of both sulindac and (-)-epigallocatechin-3-gallate has been reported [6]. The effect could be exerted through the increase of the plasma membrane receptor for lectin, which might increase tissue sensitivity to nigrin b, or alternatively be caused by polyphenol-dependent amplification of the apoptotic signals triggered by nigrin b. The present findings support the emerging belief that consuming large amounts of polyphenols may promote adverse effects when the intestines are injured. Further work will address the potential apoptotic mechanism of the observed toxic synergy between both Polyphenon 60 and nigrin b.

This study revealed that neither oral administration of large doses of GTP nor i.p. administration of sub-lethal nigrin b separately showed an effect on survival of mice. However, the oral administration of GTP to simultaneously nigrin b-treated animals was found to enhance nigrin b-dependent toxicity in mice, suggesting that GTP and/or GTP-derived active metabolites potentiate the derangement promoted by nigrin b on the small intestine crypt cells. Our results would prove useful for cancer therapy research that uses polyphenols as driving therapeutic drugs or as adjuvant supporting conventional therapy and also raise concerns on the use and abuse of concentrated green tea extracts as a food antioxidant supplement, in particular when a simultaneous toxin-driven tissue derangement occurs.

This study was supported by grants from the Regional Government of Castilla y León [Junta de Castilla y León] (GR106-Regional Education Ministry and Regional Health Ministry) and UVa-GIR, the Complutense University UCM/CAM research group 950247 as well as MAT2010-21621 C02-01 from the Ministry of Innovation and Science (Spain). We also wish to thank J.E. Basterrechea, U.L. Santiago and T. Rodríguez for technical assistance, and Philip Jaggs for manuscript correction.