Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid exchanges (SCE) and polycyclic aromatic hydrocarbon (PAH)-DNA adducts were evaluated in a group of firefighters; chromosomal aberrations and hprt mutations were evaluated in a group of cancer patients undergoing radioimmunoglobulin therapy (RIT); SCE and acrolein-modified DNA were measured in cancer chemotherapy patients and in pharmacists preparing chemotherapy prescriptions; and SCE and PAH-DNA adducts are being measured in U.S. army troops stationed in Kuwait. Our results indicate that both SCE and PAH-DNA adduct levels were not elevated in firefighters, but that other factors such as smoking status and race were risk factors for increased SCE and PAH-DNA adducts. RIT was found to increase background rates of chromosome-type aberrations and frequencies of hprt mutations and there was a strong correlation between levels of therapy-induced chromosome damage sustained in vivo and in vitro sensitivity to radiation-induced chromosome damage. Peripheral blood lymphocytes of cancer patients treated with cyclophosphamide showed higher levels of SCE and had a higher incidence of acrolein adducts in DNA. Lymphocytes from pharmacists preparing antineoplastic drugs were found to acquire increased in vitro sensitivity to SCE induction by phosphoramide mustard with increased lifetime duration of drug handling. A prospective, longitudinal study was performed to identify environmental factors that modulate genetic damage in breast cancer patients. Women with benign breast masses and no apparent disease served as controls. Mutant frequency, cloning efficiency, and chromosomal aberration frequency did not differ significantly among the three groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Induction of DNA damage and the resulting adverse sequelae such as mutations and chromosomal rearrangements are the primary mechanisms for induction of cancer. Recent studies have shown how these events are involved in activation of dominant oncogenes and the inactivation of tumor-suppressor genes (1). This information permits the design of multidisciplinary approaches for monitoring human populations for exposure to carcinogenic agents. These approaches have used the collaborative efforts of a number of disciplines including epidemiology, occupational medicine, cytogenetics, immunology, molecular biology, and statistics. This paper summarizes the results of previously published studies on groups of firefighters, pharmacists, and cancer patients. These groups have been exposed to a number of modalities including pyrolysis and combustion products, cytotoxic drugs, and radioimmunoglobulins. End points examined in these populations include chromosomal aberrations, sister chromatid exchanges, hprt mutations, and DNA adducts. The utility and disadvantages of each of these end points and potentially confounding variables are discussed.

Results and Discussion
Firefighters Firefighters are exposed to potentially carcinogenic combustion and pyrolysis products during the course of their work. A group of43 firefighters and matched controls were examined for DNA damage that may be related to occupational carcinogen exposures (2). Using peripheral blood lymphocytes, we examined a) baseline sister chromatid exchange (SCE) frequency, b) SCE induction by in vitro challenge with mitomycin C, and c) PAH-DNA adduct levels. Occupational exposures were determined from histories of firefighting activity, and the presence of confounding factors (e.g., tobacco smoking, charcoal-  (Table 3). However, smoking and alcohol consumption were positively associated with antigenicity (p = 0.09 and p = 0.07, respectively). No association was found between baseline frequency of SCE and the presence of PAH-DNA adducts.

Hepatoma Patients Treated with Radioimmunoglobulin Therapy
Lymphocytes from patients undergoing radioimmunoglobulin therapy (RIT) were examined for chromosome aberrations expressed immediately upon explant and for chromosome aberrations induced by subsequent challenge with y radiation after PHA-stimulated proliferation. In addition, hprt mutant frequencies were assessed in this same population. Primary hepatoma patients who are candidates for RIT are cyclically injected at approximately 8-week intervals with 30 mCi of 131I-labeled antiferritin. Each cycle results in a whole body radiation dose of approximately 0.3 Gy.  Chromosomal Aberrations. Lymphocytes from individual patients undergoing radiolabeled immunoglobulin therapy were examined both for chromosome aberrations expressed immediately upon explant, or for chromosome aberrations induced by a subsequent challenge of -y-rays after phytohemagglutin-stimulated proliferation (4). Despite interpatient variation, there is a strong correlation between levels of chromosome aberrations observed in the initial mitosis after mitogenic stimulation and levels induced by a challenge dose of radiation in replicate cultures after several cell cycles of growth (Fig. 1). These data indicate that even after proliferation, human lymphocytes retain a memory of in vivo exposure to ionizing radiation that can be observed by challenge with a clastogenic agent.
Hprt Gene Mutations. Gene mutations induced in vivo can be measured at the hprt locus in human T-lymphocytes. Twenty-eight patients receiving 131I and/or 90Ylabeled antiferritin were studied for mutant frequencies and compared to a control population (5,6). Major observations resulting from these studies were a) mutant frequencies for patients were increased by an order of magnitude compared to nontreated controls (68.0 x 10-6 versus 6.8 X 10 -6); b) there was a good correlation between mutant frequency and the amount of radioactivity given in the first treatment; however, there was a poor correlation between mutant frequency and the total amount of injected isotope over several cycles of treatment; c) molecular analyses of mutant clones indicated that radiation induces a higher frequency of gross structural alterations (detectable by Southern blotting) than occur in spontaneous mutations; and d) T-cell receptor analysis showed that 84% of mutants arose independently, approximately the same as in seen in control populations.

Cancer Patients Treated with Cyclophosphamide
Cyclophosphamide is a chemotherapeutic alkylating drug commonly used in the treatment of breast cancer, hematopoietic and lymphatic cancers, as well as other malignancies. Cyclophosphamide is a known human carcinogen associated with increased risk for secondary, treatment-associated cancers, most commonly myelogenous leukemias. Major metabolic products associated with cytotoxicity and carcinogenicity are phosphoramide mustard and acrolein. We studied cancer patients treated with cyclophosphamide and pharmacists involved in preparing anticancer drugs (7,8).
Acrolein Adducts. Blood specimens were obtained from 27 cancer patients who had primarily breast and hematological malignancies. Twelve patients had been therapeutically treated with a regime that included cyclophosphamide while 15 patients were newly diagnosed, untreated, and served as controls. Acrolein adducts were measured either by ELISA or immunodot blot (IDB). The ELISA assay gave positive results in 4 of 12 treated patients and in 0 of 15 nontreated patients. The IDB assay fared slightly better identifying 6 of 12 treated patients as positive and 0 of 15 untreated controls (Table 4)  SCE. Blood specimens were obtained from 39 cancer patients; 19 treated with cyclophosphamide and 20 untreated controls. Therapeutically treated patients showed statistically higher baseline SCE frequencies that controls (p < 0.016) and returned to baseline in 4-6 weeks after the last treatment dose. Cultured cells from patients treated with cyclophosphamide showed similar in vitro responses to challenge doses of phosphoramide mustard as did cells from control patients.

Pharmacists Handling Anticancer Drugs
SCE. Blood specimens were obtained from 34 pharmacy workers who had varying duration of anticancer drug handling (9). Each subject completed a questionnaire cov- ering past medical history, family history, current smoking, alcohol, diet, medication, and lifetime occupational history. The mean baseline SCE for the population was 5.19 ± 0.17 and was not correlated with duration of drug handling. However, a strong correlation was demonstrated between inducible SCE values and lifetime duration of drug handling for low-dose phosphoramide challenge (0.1 mg/mL, r = 0.63 p < 0.0001) and for high-dose phosphoramide challenge (0.25 mg/mL, r = 0.59 p < 0.0001, Fig. 2).

Women with Benign and Malignant Breast Masses
A prospective, longitudinal study was performed to identity environmental factors that modulate genetic damage in breast cancer patients (10). A total of107 women (49 with breast cancer, 52 with benign breast masses, and 6 normal women) were enrolled.
Hprt Analysis, Chromosomal Aberrations, and SCE. Mutant frequency at the hprt locus and cloning efficiency of peripheral blood lymphocytes did not differ signifilcantly among the three groups. Mutant frequency increased with age, history of cigarette smoking, and total number of years smoking. Chromosomal aberrations frequencies were similar in lymphocytes of 28 women with benign breast disease (5.3 ± 2.7 aberrations/100 cells) compared to 11 breast cancer patients (5.2 ± 2.1 aberra-  Total Exposure Hours (Log) tions/100 cells). The mean SCE incidence in lymphocytes from 23 women with benign disease was 9.3 ± 1.6, whereas the rate in cells from 8 cancer patients was 7.5 ± 1.6 (p = 0.01). However, 14 of the former group were smokers, whereas only one of the cancer patients smoked. There was no correlation between the hprt mutant frequency and either aberrations or SCE.

American froops Stationed in Kuwait
We are currently studying a cohort of 60 American troops deployed to Kuwait after Desert Storm. The U.S. government is concerned about potential health hazards associated with pollutants generated by oil-well fires. Blood samples were drawn before deployment, at one time point after 8 weeks in Kuwait, and at another 4 weeks after returning to Germany. SCE and levels of PAH adducts are currently being quantified in this longitudinal study.

Conclusions
We have used batteries of cytogenetic, biochemical, and molecular techniques to monitor diverse human populations who may be at increased risk for cancer because of exposures related to occupation, lifestyle, or medical treatment. Each end point has benefits and disadvantages. For example, induction of SCE is a sensitive end point for certain chemical exposures, especially for exposure to cigarette smoke. However, SCE frequencies respond only marginally to ionizing radiation. In addition, the significance of SCE induction is still not clear. Mutation at the hprt locus has proved to be a sensitive indicator of exposure to a variety of agents. On theoretical grounds, however, it is limited to the detection agents that induce point mutations; agents inducing primarily chromosome deletions would not be detected. Additionally, hprt mutations do not appear to occur in stem cells because mutant frequencies return to normal levels in 2-3 months after cessation of exposures. Use of antibodies against specific DNA adducts has the advantage of a high level of specificity for particular types of damages. Clearly, such an approach would not be useful for general screening. Also, because DNA adducts are repairable, they are considered as precursors to genetic events. Detection of DNA adducts using antibodies also appears more specific than sensitive (more subject to false negatives than false positives). Chromosomal aberration analysis has been the most widely used of any ofthe end points. Aberrations have been found to persist in the peripheral blood for for many years, as has been noted for the survivors of Hiroshima and Nagasaki. One must always remember, however, that the cytogenetics events that are generally scored are cytotoxic to the cells that display them and therefore are of no genetic consequence.