Workshop Overview: Hepatotoxicity Assessment for Botanical Dietary Supplements

Kristine L. Willett*,1, Robert A. Roth{dagger} and Larry Walker{ddagger}

* Department of Pharmacology and Environmental Toxicology, School of Pharmacy, University of Mississippi, University, Mississippi 38677; {dagger} National Center for Food Safety and Toxicology, Michigan State University, East Lansing, Michigan 48824; and {ddagger} National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677

Received December 6, 2003; accepted January 6, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
Botanical dietary supplements (herbal products) have flooded the market in the United States over the past decade, and studies show a significant percentage of Americans use them. With increasing frequency and duration of exposure, some serious adverse effects, though relatively uncommon, have been reported. Among the most troublesome is the association of some botanicals with serious hepatotoxicity. In some cases, hepatotoxicity has been linked to the consumption of botanicals with recognized hepatotoxic components (e.g., pyrrolizidine alkaloids). However, in other cases, the causative agent(s) is less clear and, overall, the mechanisms of hepatotoxicity are poorly understood. To help create a scientific basis for understanding botanical-induced hepatotoxicity and better tools for hepatotoxicity assessment and prediction, the National Center for Natural Product Research (NCNPR) hosted a workshop (September 8 and 9, 2003) in cooperation with the Center for Food Safety and Applied Nutrition (CFSAN) of the Food and Drug Administration (FDA). The workshop featured presentations by 22 experts and was attended by 65 individuals. The agenda can be found in the supplementary data at www.toxsci.oupjournals.org.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
The purpose of the workshop was to review and discuss methods of assessing the hepatotoxicity of botanical dietary supplements, with an emphasis on in vitro techniques that might be applied to the assessment and, eventually, prediction of hepatotoxic potential. Although application of existing and emerging methodologies can characterize and even predict some hepatic insults, the workshop identified important gaps in understanding that should be addressed by researchers.

In the United States, the market penetration of herbs, megavitamins, and homeopathic products has reached 42%, and estimates suggest Americans spend $4–12 billion on herbal products annually (Mackowiak et al., 2001Go; Zeisel, 1999Go). These products are routinely taken without medical consultation or disclosure. There are popular perceptions that, because the products are natural, they are safe and that they have been used for centuries/millennia without harmful effects. For a variety of reasons, history of use for a botanical is not a guarantee of safety, particularly with long-term use, at high doses, or with comedications and/or comorbidities.

Among the most serious safety concerns for botanical dietary supplements is the potential for liver injury. Botanical/herbal products such as kava, germander, chaparral, Ephedra, and comfrey have been associated in case reports with rare but severe cases of liver failure (Estes et al., 2003Go; Favreau et al., 2002Go; Stedman, 2002Go; Teschke et al., 2003Go). Several of these cases have occasioned considerable controversy regarding causation, but clearly there are grounds for concern that use of some botanicals is associated with hepatotoxicity. A critical need exists to develop a greater understanding of the determinants of sensitivity to botanical supplement–associated liver injury, the mechanisms involved, and how to better assess/predict the toxic potential of botanical products. Thus, the NCNPR workshop was envisioned as a forum for discussion of currently available data and research tools, emerging technologies, and research needs for the assessment of hepatotoxicity of botanical ingredients.


    REGULATORY ASPECTS
 TOP
 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
In the United States, under the Dietary Supplement Health and Education Act (DSHEA) of 1994, most herbal products are categorized as dietary supplements and, as such, cannot be sold with label claims for treatment or prevention of disease. Safety assessment requirements for dietary ingredients are quite different from those for pharmaceuticals. With the exception of new dietary ingredients, dietary supplements need neither Food and Drug Administration (FDA) approval before marketing nor the registration of the products or their manufacturers with the FDA (U.S. Food and Drug Administration, 2003Go). Under the DSHEA, the FDA cannot remove botanical dietary supplements from the market unless there is evidence of harmful effects or mislabeling. This regulatory approach is in contrast to the drug approval process in which both proven efficacy and safety must be established in humans.


    BOTANICAL SUPPLEMENTS AND HEPATOTOXICITY
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 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
The metabolic function of the liver is primarily responsible for the detoxification of structurally diverse xenobiotics, leading to metabolites that are readily excreted in bile or urine. There are also, however, numerous examples of xenobiotics for which enzymes, particularly Phase I enzymes, lead to metabolic activation of protoxicants.

Drug-induced liver injury may be manifested as acute hepatitis, cholestatis, or both, and may be further categorized as predictable (high incidence, dose-related) and unpredictable (low incidence that might or might not be dose-related). The unpredictable reactions include idiosyncratic reactions (without allergic features) and immune-mediated hypersensitivity reactions (with fever, rash, eosinophilia, and other allergic symptoms; Kaplowitz and DeLeve, 2003Go). Predictable liver toxicities are often discovered in preclinical and clinical testing for drugs. However, no such testing is currently required for botanicals marketed as dietary supplements. Furthermore, because the chemistry of these plant-derived products can be quite variable and the use sometimes sporadic, even the occurrence of predictable hepatotoxicities may be rare. Unpredictable hepatotoxicity associated with either drugs or botanicals is also extremely difficult to recognize or assess, because the prevalence of adverse reactions ranges between 1 in 10,000 and 1 in 100,000 against a large background (Hampson and Harvey, 2002Go). Regardless of etiology, hepatotoxicity is usually associated with inflammatory processes that involve a wide range of cell types, adaptive immune responses, repeated or prolonged exposure, and, to varying degrees, repair/regeneration processes, making the progression of injury very complicated. Recognition of causality in idiosyncratic hepatotoxicity is further confounded by environmental (underlying disease, comedication, inflammation, or tissue injury) or genetic factors (drug metabolism or transport differences), induction or inhibition of metabolizing enzymes, and the molecular nature of the toxicant (Boelsterli, 2003Go).

Herbal products that have been associated with hepatotoxicity have been the subject of several reviews (Kaplowitz, 1997Go; Pageaux and Larrey, 2003Go; Stedman, 2002Go); a partial listing is provided in the supplementary Table 1 (see supplemental material at www.toxsci.oupjournals.org). Botanicals containing pyrrolizidine alkaloids (PAs) have been recognized for their hepatotoxicity for over 70 years (Stedman, 2002Go). PAs can be found in more than 350 plant species, but are typically in plants from the Heliotropium, Senecio, Symphytum, and Crotalaria species. Human exposure typically occurs from teas, contaminated foods, or herbal preparations. Because of the PAs found in comfrey, companies have been advised by the FDA to remove comfrey products for internal consumption from the market (www.cfsan.fda.gov/~dms/dspltr06.html).

The FDA has also recently warned against the use of LipoKinetix, a dietary supplement used for weight loss (www.cfsan.fda.gov/~dms/ds-lipo.html; Novak and Lewis, 2003Go). LipoKinetix contains norephedrine, sodium usniate, 3,5-diiodothyronine, yohimbine, and caffeine. Patients taking this supplement developed heptatotoxic symptoms to varying degrees, including elevated alanine aminotransferase levels, jaundice, acute hepatitis, massive necrosis, and fulminant hepatic failure (Estes et al., 2003Go; Favreau et al., 2002Go). The active hepatotoxin(s) in LipoKinetix remains to be identified.

Not all cases of dietary supplements and their relationship to hepatotoxicity are as clear as the examples above. For instance, there is an ongoing debate about the safety of kava. Kava is a shrub native to the South Pacific; its roots have been used for generations to prepare a psychoactive beverage, and dietary supplements containing kava extracts are used as anxiolytics. Products containing kava pyrones were withdrawn in Germany in June 2002 after suggestions of hepatotoxicity. Controversy continues as patients’ case histories are reanalyzed and the regulatory decision is questioned (Schulze et al., 2003Go; Teschke et al., 2003Go).

Complexities in Chemical and Biological Characterization of Botanicals
A major theme of the workshop was the contrast between pharmaceuticals and botanical supplements with regard to chemical complexity. This complicates our understanding of many aspects of supplement use, from the basic understanding of what exactly is being ingested, to the interpretation of clinical efficacy, to the monitoring of adverse events. Furthermore, in research of the biological activities of these supplements, many factors contribute to variable results. The uncertain identities of raw materials used (misidentified species and adulterant or contaminant plants); the variability in constituents within a species, depending on plant part, developmental stage, and growth conditions; and the differences in harvest, processing, and product formulation all contribute to such variable results. Also, many products contain not just a single plant but combinations of several herbs; this is particularly common in Ayurvedic or traditional Chinese medicine remedies. The science in the field of chemical authentication of botanicals is still developing, and monitoring of quality control from this perspective is often inadequate.

Predicting Hepatotoxicity Assessment: Selecting Relevant Models
In the pharmaceutical industry, predicting idiosyncratic hepatotoxicity reactions has been a formidable challenge. In development, a pharmaceutical compound will typically be exposed to tiers of in vitro tests to assess stability, bioactivation, mutagenicity, cytotoxicity, interactions with drug-metabolizing enzymes (DMEs), or other parameters. Also, new drugs are evaluated in vivo in at least two species for a variety of safety end points including liver injury. Even with this rigorous approach, the incidence of idiosyncratic hepatotoxicity is unacceptably high. These parameters of evaluation for botanical products, for which chemical composition and dose of the offending constituent may be unclear, are especially complex. How can pharmaceutical industry approaches be applied to botanical supplements? Do in vitro models adequately predict the potential for a botanical ingredient (or a drug) to cause injury? What are the right models for study in vivo, considering species differences in bioavailability, metabolism, and susceptibility?

It has been suggested, based on preliminary findings using human liver microsomes, that kava pyrones containing a methylenedioxybenzyl moiety can be converted to reactive quinone metabolites in vitro; this might account for observed hepatotoxic effects. Studies in animal models might provide insight; however, it appears that rodents do not serve as good predictors of these effects because the demethenylation of dihydromethysticin occurs in human, but not rat, heptatocytes. In a recent report, rats did not display hepatotoxic injury with large doses of kava extracts (Singh and Devkota, 2003Go). Even when an appropriate animal species can be identified, laboratory animals are typically highly inbred, healthy, and exposed to only a single test compound at a time. In contrast, humans are genetically diverse, often take botanical products because of poor health, and are usually exposed to many other environmental factors, including possible comedications.


    Drug Metabolizing Enzymes
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 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
A primary consideration for potential safety concerns with botanicals is the area of drug interactions. Recent studies have highlighted instances of induction or inhibition of DMEs by botanical constituents (Zou et al., 2002Go). These may have undesirable effects, augmenting or impairing the bioavailability of drugs with narrow therapeutic indices or enhancing bioactivation of some drugs to reactive intermediates, with consequent toxic responses. Also, certain botanical constituents themselves may be bioactivated by DMEs to form reactive metabolites (e.g., kava).

Human hepatocytes, because of the relevant suite of DMEs, have been used to study metabolism of botanical components or to identify reactive metabolites. They are also convenient for investigating interactions with coadministered drugs (Li, 2002Go). The pros and cons of human tissue models have been the topic of a prior workshop and were reviewed previously (MacGregor et al., 2001Go). The presumed advantages of primary human hepatocytes are the following: (1) an intact cellular architecture, as compared to microsomal preparations; (2) no need for cross-species extrapolation; (3) the cells maintain relatively differentiated phenotypes compared to cell lines in culture; and (4) both induction and inhibition at the expression level can be observed. Nevertheless, a disadvantage of using primary human hepatocytes is the limited availability of tissue. Various groups have used analogous approaches with human liver cell lines. Though these may lose some of their differentiated metabolic pathways, it is possible to select sublines with desired characteristics. An example is a specially selected subline of HepG2 cells (C3A), which, unlike parent HepG2 cells, exhibits toxic responses to aflatoxin B1. With C3A, CYP3A and CYP1A induction responses are also observed (Kelly and Sussman, 2000Go).

In addition to differences in susceptibility of human and animal models, it is now well known that certain polymorphisms among humans can significantly affect susceptibility to hepatotoxic injury (Watkins, 2003Go). Polymorphisms in cytochrome P450 genes can predispose humans to fast or slow metabolism of xenobiotics, including botanical components. An illustrative example is the CYP3A-mediated metabolism of germander. Germander (Teucrium chamaedrys L.) is used as a weight control adjuvant, and it contains neo-clerodane diterpenoids that are bioactivated by CYP3A. In one study, induction of apoptosis in vitro was blocked by a CYP3A inhibitor and enhanced by CYP3A induction (Loeper et al., 1994Go). Variability in human CYP3A includes polymorphisms in CYP3A5 and CYP3A7 that occur at different frequencies in various populations. These polymorphisms can be major determinants of the percentage of the total CYP3A expressed in human liver (Burk et al., 2002Go; Kuehl et al., 1990Go). If CYP3A5 and CYP3A7 play a role in the germander CYP3A metabolism, certain individuals with polymorphisms in these isoforms could be predisposed to germander-induced hepatotoxicity. (Germander usage in France led to 26 cases of cytolytic hepatitis.) Given advances in the field of pharmacogenomics, such genetically determined variations in dietary supplement responses will be easier to recognize in the future.

Recent important advances in our understanding of the induction of CYP450s by xenobiotics emphasize the role of a variety of nuclear hormone receptors and their response elements, such as the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR), which mediate induction of CYP2B and CYP3A. St. John’s wort extracts appear to induce CYP3A4 via the human PXR. These receptors also display some species-dependent characteristics; human and rodent CAR and PXR respond differently to inducers. CAR also appears to regulate glucuronidation and glutathione S-transferase activity, and Yin Chin (Artemisia capillaris), a component of a traditional Chinese medicine remedy, appears to increase bilirubin clearance via CAR activation. An important innovation is the use of humanized rodent models (Huang et al., 2003Go). Mice can be genetically modified to contain human nuclear receptors or human cytochrome P450 genes. These models may then provide better representations of how botanicals will interact with these human proteins, while still maintaining the advantages associated with rodent toxicological models (Huang et al., 2003Go). It is noteworthy that such studies have demonstrated that CAR-humanized mice (with murine CAR replaced by the human form) respond to Yin Chin by increasing bilirubin clearance.


    Involvement of Multiple Cell Types and Mediators
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 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
Along with the uncertainties introduced with species variability and genetic polymorphisms in human populations, other factors complicate the ability to predict botanical hepatotoxic responses and to study their mechanisms. Although approaches in vitro are typically used in toxicity testing and these isolated systems are useful to identify potential mechanisms of action, how well cell-based models represent the situation in vivo is an area of concern. For example, although some hepatotoxicants are certainly directly toxic to hepatic parenchymal cells in vitro, in many cases other cell types have been implicated, including Kupffer cells, neutrophils, T-lymphocytes, stellate cells, and sinusoidal endothelial cells.

Monocrotaline (MCT), a pyrrolizidine alkaloid, causes pronounced oncotic necrosis of hepatic parenchymal cells following an acutely toxic dose to rats. Recent research indicates that the toxic response requires more than just MCT and hepatic parenchymal cell interaction. MCT-induced injury to sinusoidal endothelial cells occurs (Copple et al., 2002aGo) and is associated with activation of the coagulation system, deposition of fibrin in the sinusoids, and centrilobular hypoxia (Copple et al., 2002aGo, 2004Go). Moreover, prevention of fibrin deposition using anticoagulant therapy protects against hepatocellular injury (Copple et al., 2002bGo). Although hepatotoxic doses of MCT do not require inflammation to cause liver injury (Copple et al., 2003Go), otherwise nontoxic doses of MCT become toxic in the presence of modest, coexisting inflammation (Yee et al., 2000Go). When rats were treated with doses of lipopolysaccharide (LPS) sufficient to induce a modest inflammatory response, subsequent administration of subthreshold doses of MCT elicited pronounced hepatotoxicity. This synergistic reaction was not observed in isolated hepatocytes exposed to MCT and LPS. The enhanced sensitivity in vivo requires Kupffer cells, neutrophils, and tumor necrosis factor-{alpha}, as well as an activated coagulation system (Yee et al., 2002Go, 2003aGo,bGo; supplementary Fig. 1 at www.toxsci.oupjournals.org). This example suggests that the contributions of various nonparenchymal cell types found in the liver, and the inflammatory mediators they produce, should be considered in the assessment of hepatotoxic potential of botanical supplements. Moreover, it raises the possibility that consumers of PA-containing products who experience an infection or inflammatory episode may be at greater risk for developing liver injury. It also raises concern that such interactions may be applicable to other classes of hepatotoxic agents present in some dietary supplements.

Emerging Technologies in Predicting Hepatotoxicity
New approaches to predicting hepatotoxic injury and understanding the mechanisms involved will undoubtedly evolve from several emerging technologies. At the workshop, speakers addressed how transcriptomics, proteomics, and metabonomics are being used in drug development and liver toxicity assessment. The validity and utility of these technologies depend on finding gene, protein, or metabolite formation patterns that are consistent with chemical/drug/herbal toxicity.

Genomic transcript profiling has been applied to understanding cellular responses to liver injury, with the aim of assembling data on hundreds of compounds, toxic and nontoxic, under one set of experimental conditions. In one approach, genes from rat liver tissue selected for their potential toxicological relevance are profiled, and the data are compared with histopathology and clinical chemistry changes. This approach is being extended to a human toxicogenomics database, though the number of compounds assessed to date is limited. At the National Center for Toxicogenomics (NCT at NIEHS), gene expression profile changes caused by acute, subchronic and chronic exposures to hepatotoxicants (peroxisome proliferators, phenobarbital, and acetaminophen) are being assessed in microarray studies. These exposures can be shown to elicit up- or downregulation of class-specific genes, consistent with known or suspected mechanisms of action.

The NIEHS is also conducting proteomic studies using the same animals and treatments as the NCT program. Through two-dimensional gel electrophoresis and mass spectrometry identification, altered hepatic gene transcripts and protein profiles are compared to determine pathways of activation or repression associated with each particular toxicant (Merrick and Tomer, 2003Go; Ruepp et al., 2002Go). In an interesting twist on the applicability of proteomics, serum proteins are separated and identified in an attempt to develop relevant biomarkers of disease and organ toxicity.

The methodology of metabonomics complements transcriptomic and proteomic approaches by using urine or plasma to analyze time-related, multiparametric metabolic responses in exposed organisms. Because this is a noninvasive technique, many metabolic pathways and the functional integrity of the organism as a whole can be tracked prior to toxicant exposure and throughout toxicity onset and recovery. Members of industry and academia are working together to develop a comprehensive metabonomics database using parallel studies of a panel of known toxicants. Proteomic and metabonomic approaches are particularly well suited for identifying toxicant-specific biomarkers of exposure that eventually could be used clinically. Currently, the technologies of transcriptomics, proteomics, and metabonomics are still being explored, optimized, and validated using drugs and toxicants for which much is known about their mechanisms of action. Once these response profiles and bioinformatics are established, the technologies will be useful to further understand and predict hepatotoxicities associated with dietary supplement use.


    SUMMARY
 TOP
 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
The occurrence of botanical-induced hepatic injury, while relatively uncommon, is a serious problem that is very difficult to manage, based on the following factors: (1) the use of botanical supplements is extremely widespread (and often unreported), and, based on the common perception of safety, supplements may be taken at high doses and over a long period of time; (2) the complexity and variability of botanical products; (3) the lack of rigorous quality controls in manufacturing; (4) limited statutory regulation of dietary supplements under DSHEA; (5) the inherent difficulty in establishing causation with adverse events; and (6) variations in susceptibility due to genetic or pathophysiological factors or coexposures to other agents that impact susceptibility.

Many of these issues were addressed in the workshop, and several critical needs were identified. Firstly, better manufacturing controls would minimize the incidence of hepatotoxic and other adverse events as well as facilitate the tracking of offending agents in cases that do occur. The proposed FDA rules for Good Manufacturing Practices (www.cfsan.fda.gov/~dms/dscgmps3.html), once implemented, will be an important first step in this direction. Secondly, more awareness and comprehensive use of avenues for adverse event reporting (e.g., FDA’s MedWatch program for health care professionals [www.fda.gov/medwatch/index.html]) will facilitate the identification of problem plants and their toxic constituents. Thirdly, more suitable screening methods for predicting hepatotoxicity need to be established so botanicals and their derived products can be reliably assessed, ideally prior to marketing. The availability of such methods will, of course, depend on extensive research to develop and validate them.

It seems unlikely that a single screening approach will be able to predict hepatotoxic potential since the mechanisms of action involved are so varied and complex. However, the development of a battery of tests, based on the thorough characterization of known hepatotoxic botanicals and correlation to liver injury in vivo, might be possible. These tests might be used to screen against the major categories of offending agents. Toward this end, research efforts are required on several fronts.

Transport/Metabolic Enzyme Interactions
Systematic evaluations of the impact of botanical products on DMEs should be undertaken, including studying the inhibition/induction of various Phase I and Phase II enzyme systems from the gut epithelium and liver. A battery of in vitro tests might allow the development of a database including this information. One problem is establishing the applicability of such tests to human consumption. What concentrations for the cellular or enzyme testing are relevant for oral bioavailability or hepatic exposure in vivo?

Related to the question of DME interactions is the potential conversion of botanical constituents that may be substrates for CYP450s to intermediates that cause liver injury. Research is needed on the identification of the specific isoforms that mediate such conversions and how these may vary in human populations to determine susceptibility to these agents.

Toxicity to Hepatic Cells
Screening for hepatotoxicity in human primary hepatocytes appears to offer distinct advantages over established cell lines (see above). However, the limitations of tissue availability and intersubject variability mitigate against widespread application. Development and use of stable hepatocyte cell lines expressing representative human DMEs would provide a valuable tool for screening. Mechanistic characterization of known plant-derived hepatotoxins could allow the design of specific screens for detection of a particular activity profile. Hopefully, the toxicogenomic or toxicoproteomic profiling with model hepatotoxins will point to specifically designed cell culture systems for detecting such patterns of injury.

Because numerous cell types interacting in complex ways mediate toxic responses for many chemicals, current in vitro systems cannot be expected to predict all responses that occur in vivo. Systems designed to allow for interactions of different cell types or mediators may be required for some modes of injury. For example, liver slice systems might be used to assess contributions of vascular or stellate cells to parenchymal cell damage. Cocultures of activated inflammatory cells or coincubation with immune/inflammatory mediators may help to simulate some of the complex interactions that can be critical in vivo for the expression of hepatotoxic injury.

Validation in Animal Models or from Human Exposure Data
For all of these in vitro tests, the correlation of findings to appropriate animal models or, where possible, to humans will be critical. This type of work has already been implemented for pharmaceuticals in some of the toxicogenomics database development and can be implemented systematically with other assay systems. It seems likely that correlations must first be developed using plant-derived pure chemical entities known to cause injury. In this way, the models could be validated and then serve as tools for working back to the plant and plant-derived commercial products.

The research recommendations we have described will require an intensive effort, and, optimally, coordinated studies and cross-validation in multiple laboratories. It seems unlikely that the botanical supplement industry will undertake such efforts if not required to do so. Even then, costs may be prohibitive for the developmental work. If such work can be funded by the relevant agencies, including FDA/CFSAN, National Center for Complementary and Alternative Medicine, Office of Dietary Supplements, and others, the screening tools thus derived could be used to bring a higher level of safety assurance for botanicals.


    SUPPLEMENTARY DATA
 TOP
 ABSTRACT
 INTRODUCTION
 REGULATORY ASPECTS
 BOTANICAL SUPPLEMENTS AND...
 Drug Metabolizing Enzymes
 Involvement of Multiple Cell...
 SUMMARY
 SUPPLEMENTARY DATA
 REFERENCES
 
Supplementary Table 1, a partial listing of herbal products that have been associated with hepatotoxicity, is available online at www.toxsci.oupjournals.org. Supplementary Fig. 1. Supplementary Fig. 1 depicts involvement of multiple cell types and mediators involved in monocrotaline-induced hepatoxic injury. The workshop agenda is also available.


    NOTES
 

1 To whom correspondence should be addressed at Department of Pharmacology, University of Mississippi, 315 Faser Hall, Box 1848, University, MS 38677. Fax: (662) 915-5148. E-mail: kwillett{at}olemiss.edu


    REFERENCES
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