Use of botanicals in food supplements. Regulatory scope, scientific risk assessment and claim substantiation. 2005.

BACKGROUND/AIMS: In the European Union, an elaborate legal framework regulates botanical products both under food and medicinal law. The decision as to which legal framework applies to an individual product may differ between the Member States. In the case of botanical food supplements, all food law provisions apply to their manufacturing, composition and marketing, including the new claims legislation. METHODS: Elements from EU and national law, scientific and other publications are brought together to investigate how to clarify the differentiation between the use of botanicals for medicinal and health-promoting purposes on a scientific basis. RESULTS: Guidance on the safety assessment and quality evaluation of botanicals is proposed in light of the different approaches described in the scientific literature with particular attention to the concept of long-term use as an integral part of safety evaluation. Guidance on claims substantiation is also included, taking into consideration the proposed legislation, the concept of long-term experience and grading of evidence. CONCLUSIONS: A model for safety and efficacy assessment of botanical food supplements in the EU is proposed, and should be taken into consideration in the development of legislation and scientific research on botanicals.

Modulation of proliferation and tumorigenic potential of cervical-carcinoma cells by the expression of sense and antisense p53.

Previous studies from our laboratory demonstrated that overexpression of wild-type p53 suppresses the growth and alters the differentiation pattern of HPV-immortalized keratinocytes (Woodworth et al: Cell Growth Differ 4: 367-376, 1993). In the present report we describe the use of sense and antisense p53 retroviral vectors to modulate the expression levels of the p53 tumor suppressor gene in cervical carcinoma cells. Representative cell lines (C-4 I, ME-180 and C-33 A) were selected for this study on the basis of their various expression levels of either wild-type or mutated p53, and also of their content of integrated human papillomavirus sequences. Our results show that p53 overexpression from the sense construct decreased the growth rate and tumorigenic potential of the HPV-negative C-4 I cell line, whereas it had no effect on the HPV-positive ME-180 and C-33 A cells. However, examination of the individual colonies early after transfection with sense constructs by light microscopy showed that overexpression of wild-type p53 in ME-180 induced significant morphological alterations resulting in cellular senescence. Nevertheless, the p53 overexpressing, ME-180-derived cell line established after selection did not exhibit any major changes compared to the parental cell line. In the case of C-33 A, the presence of high levels of mutant p53 prevented the exogenous wild-type p53 from causing any significant modulatory action on their proliferation or neoplastic phenotype.

Extrapolation of animal toxicity to humans: interspecies comparisons in drug development.

Different methods for converting the dose-related toxicity of drugs from animals to humans are reviewed. Each method is analyzed with respect to its utility and limitations. Linear extrapolations from animals to humans based on body weight equivalence are shown to be inaccurate unless species-specific conversion factors are used. Extrapolations based on surface area equivalence are more accurate, do not require conversion factors, and may be used when pharmacokinetic data are not available. Ultimately, interspecies conversions are most reliable when pharmacokinetic data are available, assuming that toxic responses are comparable among species for similar blood levels. Two pharmacokinetic-based approaches may be used: direct use of plasma concentration or area under the concentration-time curve (AUC) and physiologically based pharmacokinetic (PBPK) models.

Synergistic effects on the initiation of rat liver tumors by trans-4-acetylaminostilbene and 2-acetylaminofluorene, studied at the level of DNA adduct formation.

Both trans-4-acetylaminostilbene (AAS) and 2-acetylaminofluorene (AAF) exert tumor-initiating activity in rat liver when administered in the initiation phase of an initiation-promotion experiment. The effects are more than additive when the compounds are sequentially combined in the initiation phase of such an experiment, and this synergism is more pronounced when AAS is given first, followed by AAF, than vice versa. In order to determine the role of target DNA dose, [3H]AAS and [14C]AAF were administered to female Wistar rats adhering to the protocol of the initiation phase of the above-mentioned experiment and the following parameters measured at the end of this phase: total radioactivity in tissues, binding to DNA, RNA and proteins in liver, adduct pattern in liver DNA and RNA. In neither combination were these parameters significantly different from those in the appropriate controls in which only one of the compounds was administered. This result indicates that combining the substances did not alter the pharmacokinetics of the individual compounds and that the target dose is additive. This suggests that effects unrelated to DNA binding, possibly promoting effects, may cause the more than additive generation of preneoplastic lesions in rat liver.

Induction of single-strand breaks and interstrand cross-links in liver DNA after the administration of 2-acetylaminofluorene and trans-4-acetylaminostilbene to rats.

2-Acetylaminofluorene (AAF) or trans-4-acetylaminostilbene (AAS) was orally or intraperitoneally administered to female Wistar rats. DNA from liver cells was analyzed for single-strand breaks by the alkaline elution assay. Only borderline effects were observed with doses (100 mumol/kg) used in animal carcinogenesis experiments. Even high doses of AAF (1,000 mumol/kg) were not effective. Methyl methanesulfonate (MMS) in vivo and gamma irradiation in vitro were shown to produce dose-dependent DNA single-strand breaks (positive control). Only a marginal effect was obtained with 100 mumol/kg MMS. The elution rate of DNA was increased by a factor of 34 in liver cells in vitro with 400 rad of gamma irradiation. Only a fraction of this rate could be demonstrated immediately after irradiation in vivo, and no lesions were found two hours later. This strongly indicates the rapid repair of single-strand breaks. Additional experiments showed that AAS, a nonhepatocarcinogen, produced more interstrand cross-links in the rat liver DNA than did AAF.