# Presentation: 539365

## Biomarkers and Species Comparisons (Metabolism & Metabolomics) 

**Susan Sumner, PhD**
**RTI International**
**National Academy of Sciences**
**BOSC Risk Assessment Workshop**
**February 2005**

## Species Comparison Issues

- Is the animal model relevant to humans
- Do you expect the effect generated in rodents to occur in humans?
- Does it exhibit a  similar  dose-response?
- Can you extrapolate from a high dose in rodents to a low dose in humans?
- How  do small “animal numbers” translate to large human populations?
- What are the pharmacokinetics and pharmacodynamics?

## BIOMARKERS CONTINUUM

- Markers of Exposure

- Markers of Effect

- EXPOSURE

- INTERNAL
- DOSE

- BIOLOGICALLY EFFECTIVE DOSE

- EARLY
- BIOLOGICAL
- EFFECT

- CLINICAL
- DISEASE

- SUSCEPTIBILITY

- Cytochrome P450
- GSTs
- N-Acetyltransferases
- Oncogenes
- Tumor Supressor Genes
- DNA Repair

- Chemicals or
- metabolites
- In blood or
- Tissues

- AUC
- DNA Adducts
- Protein Adducts

- DNA Strand Breaks
- SCEs
- Micronuclei
- Chromosomal
- Aberrations
- Mutations
- Oncogene Activation
- Tumor Suppressor Gene Mutations

- Fennell 1996

- Adapted from NRC 1989

## Biomarkers & Species Comparisons

- Tissues and biological fluids from rodent dose-response studies may be used to develop markers of exposure and effect.
- Urine and blood based markers have potential for use in human-to-animal model comparisons.
- Urine markers provide an integrated read-out of events that have occurred over time.

## Biomarkers and Metabolism

**Metabolizing factors are known to be species and strain specific; necessitating the use of global detection methods to access the human relevancy of markers developed in animal models. **
**Elucidation of **_**xenobiotic**_** metabolism and alterations in  **_**endogenous**_** metabolism as a function of chemical exposure, dose-level,  and response provides one means to develop the causal relationships between metabolites, pathways, and effects**.

## Global vs Targeted Methods

**Conventional Methods are Directed:**
**Look for what you already know about**
**In cross species studies, “new or additional responses” may not be noted because methods are selective or not well resolved.**
**Need methods to find out what you do not know**
**Non-Directed Methods : For metabolism...**
**LC-TOF-MS**
**NMR**
**Incorporation of carbon-13 labeled xenobiotics in study design**

## 13C1 13C NMR and Chemical Metabolism  

**Sumner et al. 1992, Chem Res Toxicol  **

- control

- 13C-AM

- Sumner et al., 2003, Chem. Res. Toxicol.

## [13C]AM Metabolism in Mice, Rats, and Man

- [13C]AM Metabolism in Mice, Rats, and Man

- Fennell et al, 2005; Toxicol. Sci., in press

## Implications of Non-Directed Approaches: More Informed Species Comparisons!  

- Mouse          Rat          Human
- Qualitative and quantitative species differences were resolved that conventional methods “missed”.
- co-migration of metabolite standards that result in erroneous findings due to similar structural features for metabolites that arise from different pathways.
- New metabolites and new pathways for the xenobiotic metabolism were discovered that had not  previously been postulated.
- “Controversial” metabolites or pathways were either confirmed or denied.

## LC-TOF-MS and Drug Metabolism

- Plumb et al. (2003) Use of LC-TOF-MS and multivariate statistical analysis shows promise for the detection of drug metabolites in biological fluids. Rapid  Commun Mass Spectrom. 17:2632-8
- ----------------------------------------------------------------------------------------
- Rats were administered vehicle or dose levels of a candidate pharmaceutical.
- Collected urine was diluted and injected on the LC-TOF-MS system and all peak intensities were normalized to the sum of peak intensities.
- Statistical methods were used to determine signals which differentiated urine samples based on dose.
- The authors  identified drug metabolites with no  priori knowledge of the compound administered.

## 7-HEG in Mice Exposed to 100 ppm EO

**7-HEG in Mice Exposed to 100 ppm EO**

- Walker et al. 1992, Cancer Res.

## Phosphotriester Adducts and DNA Strand Breaks

**Phosphotriester Adducts and DNA Strand Breaks**

- Yates et al., 1993, Carcinogenesis

## Recommendations

- How big is our light bulb?
- Require  incorporation of high resolution and non-selective analytical approaches  for  biomarker analysis with consistency across species. Not just the historical or comfortable approach.
- 14C[X] and chromatography will not necessarily provide the resolution for characterization & species comparisons.
- .
- Careful use of published biomarker data, particularly when different analytical techniques have been applied to study a species or more than one species.

## Omics

- Omics technologies help us move away from the lamp post effect.  However.....
- GEP profiling is only as good as the selection of genes on the chip.
- Proteomics  is only as good as the gel resolution or  identification methods.
- And metabolite profiling picks up only what the specific analytical method measures.
- We can now look out  wider windows & with more light.
- But...are we looking out the right window (s)?

## METABONOMICS

- The quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification*.
- Applies to cells, organs, biofluids, breath.
- Assessment of changes in endogenous metabolites may help to define patterns to associate with “critical events”.
- The pathway relationship of these markers may identify mechanisms of injury or susceptibility.

- *Jeremy Nicholson, 1999

## Sigma Metabolic Pathways Chart

- Sugars

- Lipids

- Steroids

- Aromatic
- amino acids

- Purines &
- pyrimidines

- Amino acids

## Metabol(n)omics: Process

- Analytical Methods to capture metabolite signals: NMR, LC/MS,  GC/MS (TOF).
- Chemometric and statistical analysis
- Separation of groups by dose, time, on set of or recovery from “disease”.
- Determining the pattern(s) of signals that are responsible for association of metabolite profile and “outcome”.
- Metabolite assignment and biochemical pathway relevance.

## Drug-Induced Liver Injury

- RTI NMR-Based Metabonomics: Jeffcoat, Burgess, Bobashev & NIEHS

## Metabolomics & Exposure to Disease Assessment

- A major benefit of metabonomics over other “omics” is the ability to analyze urine and blood from humans.
- For populations in which exposure or disease are well defined, metabolomic profiles developed for urine or blood may result in patterns that can be clearly associated with exposure level or outcome.
- Diseases or exposures that result in alterations in endogenous profiles that are well defined from control populations will be more readily associated with specific metabolic perturbations.

- Slide 1

## Exposure to Disease  

- Because environmental or potential industrial exposures are typically  to low levels of chemicals, the inherent variability in human samples may in fact mask defining the relationship between exposure and outcome.
- It is likely that  controlled studies in humans will help to define the effect of low level exposures on perturbations in endogenous components.
- Dose-response studies in animal models can be used to understand MOA and then to extrapolate profiles to evaluate human data (e.g., develop corrollarry markers using tissue and urine from rats for assessment of human urine)
- Mapping to biochemical pathways will be critical in developing markers that can cross species.

- Slide 2

## Metabolism of [13C/ 14C]-HCP

##  13C-NMR Spectra:HCP-Induced Changes in Endogenous Metabolites

- Control Urine

- HCP Urine

## Synthesis of Ascorbic Acid by the Rat

**Synthesis of Ascorbic Acid by the Rat**

## Metabolism of Methoxyethanol

**Metabolism of Methoxyethanol**

## Expected Effects of Chemical Exposure on Endogenous Metabolism

- Xenobiotic metabolism – demand for NADPH, GSH, UDPGA, PAPS, etc., and  up/downstream ripples.
- Entry of xenobiotic into endogenous metabolism (e.g. ethanol, acetate-esters)
- Enzyme Induction
- Targeted enzyme inhibition as a therapeutic mode of action.
- What is an occurrence and
- what is predictive of adverse outcomes?

## Metabol(n)omic Issues

- How conserved are mammalian pathways, enzymes, and regulation of flux?
- What is the role of diet, diurnal variation, gender, age, seasonal variation, and strain/ethnic origin?
- What is the conservation of (compartmentalization) of metabolites across cells, tissue, and bio-fluids?

## Recommendations

- Metabol**omic **assessments will benefit from:
- samples from relevant controlled  human exposures
- long-term population monitoring with serial metabolite **profile generation** and long-term disease association
- development of databases to define influence of dietary intake, drugs and chemicals, life-style factors and susceptibility issues on endogenous **profiles**
- associated databases from animal studies (models for onset of and recovery/repair from specific diseases or insults)
- development of methods for extrapolation of metabol**omics** data through pathway analysis