Measurement of micronucleated erythrocytes and DNA damage during chronic ingestion of phenolphthalein in transgenic female mice heterozygous for the p53 gene.
Phenolphthalein, a common ingredient in nonprescription laxatives and a multisex, multispecies rodent carcinogen, was evaluated under chronic exposure conditions for genotoxicity in transgenic female mice heterozygous for the p53 gene (heterozygous TSG-p53 mice). Phenolphthalein was administered in the diet at 200, 375, 750, 3,000, and 12,000 ppm (corresponding to a time-weighted average of 37, 71, 146, 569, and 2,074 mg/kg/day, respectively) for 6 months (183 days). On days 39, 92, 137, and 183 of treatment, peripheral blood samples were collected and evaluated for the frequency of micronucleated polychromatic and normochromatic erythrocytes (MN-PCE and MN-NCE, respectively), the percentage of PCE (%PCE) among total erythrocytes, and the extent of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in leukocytes. In addition, the extent of DNA damage was evaluated in liver parenchymal cells sampled from mice at the end of the 6-month treatment period. DNA damage was evaluated using the alkaline (pH > 13) Single Cell Gel (SCG) assay. In addition, using a modified SCG technique, the frequencies of leukocytes and liver parenchymal cells with extremely low molecular weight DNA (indicative of apoptosis and/or necrosis) were determined. At each sample time, phenolphthalein induced a highly significant, dose-dependent increase in the frequency of MN-PCE and MN-NCE and in %PCE. Maximal induction of MN-PCE and %PCE decreased with increasing treatment duration, most likely due to a treatment duration-dependent decrease in the relative amount of ingested phenolphthalein. A comparative analysis of the kinetochore status of MN in erythrocytes sampled from control mice and mice ingesting phenolphthalein at 12,000 ppm for 183 days indicates that the induced MN resulted predominantly but not exclusively from numerical chromosomal damage. The analysis for increased levels of DNA damage in blood leukocytes was inconclusive, with a small but statistically significant increase in DNA migration on days 39 and 137 but not on days 92 and 183. The extent of DNA migration in liver parenchymal cells sampled from mice at the end of treatment was not altered significantly. The frequencies of apoptotic and/or necrotic leukocytes and liver parenchymal cells were not increased among mice ingesting phenolphthalein. The lowest effective dose at which a significant genotoxic response (i.e., the induction of MN-NCE) was detected was 200 ppm, the lowest dose tested in this study. This dose in mice is comparable to doses (on a mg/m2 basis) experienced by humans.
Tice
R R
RR
Integrated Laboratory Systems, Research Triangle Park, North Carolina, USA. rtice@ils-inc.com
Furedi-Machacek
M
M
Satterfield
D
D
Udumudi
A
A
Vasquez
M
M
Dunnick
J K
JK
D000284:Administration, Oral; D000818:Animals; D002400:Cathartics / Q000008:administration & dosage*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects*; D004032:Diet; D004913:Erythrocytes, Abnormal / Q000737:chemistry / Q000187:drug effects* / Q000648:ultrastructure; D005260:Female; D016158:Genes, p53 / Q000187:drug effects* / Q000235:genetics*; D006579:Heterozygote; D018386:Kinetochores / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D048629:Micronuclei, Chromosome-Defective / Q000737:chemistry / Q000187:drug effects; D015162:Micronucleus Tests; D020113:Phenolphthalein; D010635:Phenolphthaleins / Q000008:administration & dosage*
D002400:Cathartics; D010635:Phenolphthaleins; D020113:Phenolphthalein; D004247:DNA