Low-level biological dosimetry of heterocyclic amine carcinogens isolated from cooked food.

The bioavailability and the bioreactivity of the carcinogenic heterocyclic amine [2-14C]2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) have been investigated at a dose approximating that likely from the human diet by accelerator mass spectrometry (AMS). [2-14C]PhIP was administered to mice at a dose equivalent ot the consumption of two 100 g beef patties (41 ng/kg). The biological half-life of PhIP was 1 hr, with 90% of the dose being excreted via the urine. Peak tissue PhIP concentrations were reached within 3 hr, with the highest levels in the tissues of the gastrointestinal tract, followed by the liver, kidney, pancreas, and thymus. Since the detection limit by AMS is dependent on the natural abundance of 14C, we have achieved further increases in sensitivity by producing mice that have 20% of the natural abundance of 14C. Use of these 14C-depleted animals allows measurements to be made near the natural level of exposure for many environmental carcinogens. PhIP-DNA adduct levels have also been measured by 32P-postlabeling at doses of 1.0, 10, and 20 mg/kg. The highest adduct levels were found in the pancreas, thymus, heart, and liver and increased linearly with dose. The principal adducts are derived from guanine.


Introduction
Determining the cancer risk that chemicals pose to humans has proven to be a difficult task. Biomarkers play an integral role in this process because theoretically they indicate progress in the development of disease, but the biomarkers must be selected to validly represent the disease mechanism (1). Fundamentally at issue is the relationship between the delivered dose and the innate ability ofan organism, both on a systemic and cellular level, to cope with the challenge. In a practical sense, translating the carcinogenic response from high-dose animal studies to lowdose human exposure scenarios is the immediate concern from the standpoint ofa) developing valid biomarkers ofexposure and effect, b) for understanding mechanisms, and c) for risk assessment.

Heterocyclic Amines in the Diet and the Need for Low-level Dosimetry
Nowhere is this risk evaluation problem more acute than in accessing the dosimetry ofthe genotoxic dietary heterocyclic amine carcinogens formed during the cooking of meat [discussed in 'Biomedical Sciences Division, Lawrence Livermore National Laboratory, Livermore, CA 94550.
Address reprint requests to K. W. Turteltaub, Biomedical Sciences Division, L-452, Lawrence Livermore National Laboratory, Livermore, CA 94550. detail by Felton and Knize (2)]. Daily human exposures for these compounds are probably on the order of a few nanograms/ kilogram ofbody weight (assuming an average 70 kg adult consumes 200 g ofmeat/day), while the animal bioassays have been carried out at doses of mg/kg (3). Likewise, pharmacokinetic, metabolic, and macromolecular adduct measurements have all been made at doses in excess of 104to 106-fold greater than human dietary exposure levels. To determine the feasibility of biomonitoring and for selecting appropriate biological exposure markers for use at human exposure levels, it is necessary to understand the pharmacokinetics, metabolism, and dosimetry of these carcinogens at relevant dietary levels and to reconcile these results to the high-dose studies at which bioassays have been traditionally carried out.
Our approach to this problem has been to focus on the DNA adduct as a potential biomarker for both exposure and effect, and we are using the high sensitivity offered by accelerator mass spectromery (AMS) for assessing adduct dosimetric relationships. AMS is a low-energy nuclear physics technique developed for measuring cosmogenic isotopes such as 14C and is ideal for studying the low-level dosimetry of isotopically labeled xenobiotics (4,5). AMS isolates and counts specific nuclei as opposed to measuring atomic decay. Thus, AMS methodology results in a 106-fold improvement in isotope detection efficiency compared to scintillation counting for 'C and has allowed us to quantitatively measure DNA adduct levels on the order of 10 -1I to 10 -12 adducts/nucleotide (6). We also have applied the 32p_ postlabeling assay to mechanistic studies of the effects of these carcinogens on DNA. The postlabeling assay is a highly sensitive 1TURTELTAUB ETAL.  (6) mice raised on a diet depleted in "4C (U) show an 80% reduction in 14C. This will allow an additional 5-fold increase in the detection of macromolecular damage. Also shown is the instrument background for 14C. Data are reported as the ratio of "C atoms to total carbon atoms isolated from the samples.
Methods C57BL/6 male mice were obtained from Simonsen Laboratory (Gilroy, CA) at 6-8 weeks of age and allowed to acclimatize for 1 week. For determination of the distribution and fate of PhIP, the mice were administered [2-14CT-labeled PhIP (50 mCi/mmole) by stomach intubation at a dose of 41 ng/kg body weight. Animals were sacrificed at time 0, 0.5, 1, 3, 6, 12, 24, 48, and 96 hr following exposure, and tissues were immediately removed. Urine was collected over ice at the same time points and pooled with urine removed from the bladder. Feces were likewise collected and homogenized in water/methanol (1:1). All samples were stored at -20°C until use. Three independent replications of the experiment were carried out. Sample preparation for AMS and 14C determinations were carried out using our previously described methods (7,8).
-24t 48 72For assessing PhIP DNA adduct dosimetric relationships, 24 48 n PhIP was administered to (6-8-week-old C57BL/6 male mice by gavage at doses of 0, 1.0, 10.0, and 20.0 mg/kg. Tissues were sentative tissues. Shown removed 24 hr later and stored at -20°C until use. In addition, 2'er (nt, and kidney (shD). deoxyribobonucleoside-3 '-monophosphates were incubated with N-hydroxy-PhIP in the presence of mouse hepatic cytosol as described in Buonarati et al. (9). DNA from the tissues and in vitro incubations were isolated and 32P-postlabeled as described previously (9). assay for DNA adduct detection that includes chromatographic separation ofspecific adducts and thus is quite useful for studying mechanisms of adduct formason and repair. Here we describe the fate and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-bipyridine (PhIP) at a dose equivalent to the consumption of200 g cooked meat and on the formation ofPhIP adducts at higher doses.

Results
PhIP was rapidly absorbed at the 41 ng/kg dose and maximal blood levels were reached within 0.5 hr (Fig. 1). Excretion was equally rapid. Ninety percent of the administered dose was eliminated via the urine and 10% via the feces over the 96-hr period. The systemic half-life of the compound was 1  Maximal concentrations of PhIP were attained in the tissues between 0.5 and 3 hr after exposure, shown in Figure 2. The highest tissue levels of PhIP or its metabolites were found in the stomach and intestine, followed by the liver, kidney, and pancreas. Some of the PhIP Ibund associated with the stomach may have been due to adsorption ofthe unabsorbed compound from the lumen surface of the tissue. PhIP levels in the tissues 96 hr after exposure were 0.5-2% of the administered dose. The residual levels of PhIP in the tissues were due possibly to covalent binding of active metabolites to DNA and protein and are currently under study.

Sensitivity of AMS
The absolute sensitivity limit for biological specimens is imposed by the natural 14C abundance present in all biological material (Fig. 3). The absolute detection limit ofthe instrument for 14C is 1 part 14C/1015 total carbon. The natural abundance of 14C in biological matter is 1 part 14C/1012 total carbon, which is two orders ofmagnitude above what can be routinely measured by AMS. In an effort to further increase our ability to measure macromolecular damage at low dose, we have attempted to reduce the amount of 14C present naturally in our animal models. Mice fed on a diet containing yeast protein obtained from organisms grown on petroleum-derived methanol have 20% of the 14C present in contemporary biological matter. This decrease has been achieved within one generation of feeding the special diet and will result in a 5-fold increase in the sensitivity of biological AMS measurements. Low-dose DNA binding analysis with heterocyclic amines is currently underway using these animals.

PhIP Adduct Levels
PhIP-DNA adduct levels have been measured in the mouse by 32P-postabeling. Three adduct spots are evident in liver tissue at all doses tested, and two adduct spots were found in all other tissues (Fig. 4A). The structural nature ofthese PhIP adducts is as yet undetermined. Incubation of reactive PhIP metabolites with 2 '-deoxynucleotide-3 '-monophosphates indicate that all three adduct spots are derived from deoxyguanosine (Fig. 4B)

Conclusion
Determining the relationships between exposure, time, systemic dose, and dose to target tissues is critical for ascertaining the validity of biological markers. Here we have shown that the dietary carcinogen PhlP, at a dose equivalent to consumption of 200 g of cooked beef, is quickly absorbed and distributed to the tissues. Elimination is also very rapid. DNA adducts are present in many tissues and increase as a linear function of dose. Adduct formation at higher doses does not seem to correlate with target tissues for carcinogenesis, suggesting that adduct formation alone is not necessarily indicative of tumorigenicity. The linearity of the adduct dose-response curve reported here for PhIP and also earlier for 2-amino-3,8-dimethylimidazo [4,5-JAquinoxaline (6) suggests that no or a very low threshold exists for DNA damage and that DNA adduct levels are indicative of exposure. The levels at which DNA damage and that DNA adduct levels are indictive of exposure. The levels at which DNA damage translates into a significant biological effect need to be determined before adducts can be validated as markers for effect or risk. Further studies are in process to define the relationship between exposure, macromolecular damage, and biological effect at low doses.