The Challenge of Identification

N the foods that make up the Western diet, the most common mutagens belong to a class collectively called the amino-imidazoazaarenes (AIAs). Not all the known food mutagens are AIAs, but the commonly found ones are. As shown in Figure 2, AIA compounds have one or two aromatic ring structures fused to the imidazole ring. They also have an amino group (NH2) on the number-2 position of the imidazole ring and can have methyl groups (CH3) of varying number and location.

Figure 2. Structure of the amino-imidazoazaarenes (AIAs), also called heterocyclic amines.
(a) The imidazole ring is common
to all AIAs. Numbers show the position of atoms on this ring.
(b) In the AIAs, an amino group
and one or more methyl groups
are attached. (c) IQ, one of the
potent AIAs found in cooked meats, has two aromatic rings attached
to the imidazole ring. The mutagenic activity of the different heterocyclic amines varies by several orders of magnitude
and can be increased when
one or more additional methyl
groups are present.

Of the list of toxic substances known to be produced during cooking, the most important may well be the AIAs. Also referred to as heterocyclic amines, these compounds are potent mutagens produced at normal cooking temperatures in beef, chicken, pork, and fish when fried, broiled, or grilled over an open flame. The pan residues that remain after frying also have high mutagenic activity, indicating that meat gravies can be a source of exposure. Our research suggests that smoke from cooking muscle meats is mutagenic as well, but any such air exposure is likely to be far less than that from eating the cooked food. Other foods, such as cheese, tofu, and meats derived from organs other than animal muscle, have very low or undetectable levels of AIA mutagens after they are cooked.

Analyzing cooked foods for mutagens requires many different methods (Figure 3). The toxic compounds in food must first be chemically extracted before purification. Over the years, we and other researchers have dramatically improved on the original extraction techniques that required various acids or mixed organic solvents in multistep schemes.
We now use solid-phase extraction, which is based on a method first described by G. A. Gross in 1990.4 After homogenizing cooked food in a blender to obtain a uniform sample,
we can extract a sample quickly and efficiently by passing it through a
series of small tapered tubes containing chemically activated particles (see step 1 in Figure 3 and Figure 4). The small amounts of organic solvents that are needed during this solid-phase extraction generate a minimum of hazardous waste.

Figure 3. Some of the steps required to extract, separate, purify, and confirm the potency and chemical structure of mutagens in cooked food. These steps show a typical sequence of events during research on a given mutagen. However, the sequence shown here can vary depending on whether our objective is to study a known mutagen or to assess the properties of a new candidate. Each of the steps is described in more detail in the text.

Separation and Purification
We use high-performance liquid chromatography (HPLC) for final separation and purification of the extracted compounds in a food sample (see step 2 in Figure 3). Liquid chromatography is a standard technique in chemistry labs. In HPLC, a liquid mixture is pumped under high pressure through a long, narrow tube filled with fine silica particles. This material differentially retards the passage of different molecular components so that each one exits after a characteristic delay or retention time. Our recent solid-phase extraction method together with HPLC allows excellent quantification from small samples (about a tenth of a hamburger patty, or one bite) and a 1- to 2-day turnaround time for results.
For unknown mutagens, a separation is carried out in several stages. We obtain about 100 fractions at the final stage, where a "fraction" is one portion of the sample material that is captured in a separate vial after exiting the HPLC detector. One fraction at the final stage of separation contains as little as a billionth of the starting material. However, because the extracts from meat and other food products cooked at elevated temperatures are tremendously potent, only a very small sample is needed for the next step--testing for mutagenic potency.

Figure 4. Researcher Cyndy Salmon uses solid-phase extraction to extract a sample by passing it through a series of small cylinders containing small amounts of organic particles.

Detection of Mutagenicity
The most widely used detection method for mutagenic potency is the Ames/Salmonella mutation test,1 which is described in more detail in the box on p. 16. This test for mutagenic activity is exquisitely sensitive and relatively inexpensive. It is also convenient because each analysis requires only 48 hours, and many samples can be analyzed in parallel (Figure 5).
The essential point to remember is that the Ames test (step 3 in Figure 3) gives us a number by which we can express the mutagenic activity of a given compound or food extract. This number by itself for a single mutagen would have little meaning. However, we now have numbers for most of the known mutagens in cooked foods and for over a hundred additional mutagens from other sources, so we can compare the mutagenic activity of many different structural types. When the Ames test is used during initial screening for new mutagens and carcinogens, it serves as a guide to the chemical purification of biologically active molecules. It can also be used to test and compare the potency of newly synthesized chemicals.

Figure 5. Julie Avila, one of the researchers in the LLNL food mutagen research group, tests mutagens in cooked beef using the Ames/Salmonella test. (a) The food sample is added to a mixture containing bacteria, nutrients, and enzymes needed for metabolism, and then (b) poured onto a petri plate. (c) Close up of growing bacterial colonies (called revertants) after 48 hours. Counting the colonies gives us a number that represents the sample's mutagenic activity.

Once a mutagen has been detected, we can characterize it further through a variety of analytical methods (step 4 in Figure 3). The type and sequence of tests depend on our objective for a given mutagen (Figure 6). For example, we can routinely determine the molecular weight through mass spectrometry and study the detailed chemical composition (the number of hydrogen, carbon, and nitrogen atoms) by high-resolution mass spectrometry (HRMS). In mass spectrometry, complex compounds are broken up into ionized fragments, which are accelerated through a magnetic field until they strike a detector. Because the path of an ionized fragment through the field is determined by its inertia, we can determine the mass of the various ions by their spatial distribution on the detector. Ultraviolet absorbance spectrometry and fluorescence spectrometry are other identification methods that are often combined with chromatography.
Substantially more effort is required if we want to identify a mutagen for the first time. For an unknown compound, we first need information on the atomic composition and the position of atoms in the molecule. This work requires HRMS and nuclear magnetic resonance (NMR) spectra (step 4 in Figure 3) together with synthesis of all possible isomers. Isomers are two molecules with the same number of atoms and molecular weight but different structures. NMR spectrometry requires the highest quantity and sample purity of all the analytical methods,
but it gives us the most definitive information on chemical structure. The exact chemical structure of a given mutagen can be proven by comparing
it with a known standard that is synthesized in the laboratory.
After the physical and chemical properties of a mutagen are known, we can use the information to determine whether that mutagen is present in other types of food. This approach gives us a way to determine the dose of a given compound in our diet and to assess the human risk associated with ingesting that compound.

Figure 6. Kathleen Dewhirst combines methods,
such as gas chromatography and mass spectrometry
or liquid chromatography and mass spectrometry, to characterize the food mutagens in
cooked meat. Mass spectrometry allows us to determine the molecular weight of a mutagen.

The Major Food Mutagens
Table 2 is a summary of the 14 major mutagens that have been identified in at least one type of heated food to date.Table 2. The presence of isomers means that we need to apply several different criteria for identification purposes because no single property, such as an absorbance spectrum, can uniquely identify all of the mutagens.
The compounds listed in Table 2 are not the only mutagens or carcinogens in food. Researchers at LLNL and elsewhere have identified other biologically active compounds, including additional aromatic amines, nitrosamines, and hydrazines. However, the heterocyclic amines we have been investigating are among the most abundant and potent substances detected to date. Because of their presence in cooked meats that are common in Western diets and their association with certain types of cancer in laboratory animals, they warrant detailed investigation.

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