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Histochemistry and Fine Structure of 4-Nitroquinoline-N-oxide-induced Nuclear Inclusions¹

Isaac Albert Research Institute of the Jewish Chronic Disease Hospital, Brooklyn, New York

Rat nephroblastoma (RNB) cells on cover slips were exposed to 200 µg % of 4-nitroquinoline-N-oxide (4-NQO) for 10 min and reincubated for 1-–24 hr. By optical microscopy within the 1st 4 hr the nucleoli became rounded and developed "caps." Azure B staining with RNase digestion revealed loss of nucleolar RNA. The residuum was composed of Feulgen-negative acidophilic protein. After 18–24 hr, tinctorially similar inclusions with surrounding clear halos were present. These were "fast green histone"-negative but gave a positive histochemical reaction for tyrosine as does the intact nucleolus.

Ultrastructurally, the nucleolus shows within the 1st 4 hr rearrangement into segments composed of aggregates of granules of differing electron density. At 18–24 hr, up to 4 frequently morphologically dissimilar bodies were found in single sections of the nucleus. Some resembled smaller remnants of the earlier nucleolar lesions. Others showed compact central cores surrounded by granule halos with intermingled dense-staining plaques or lay in loosely arranged fields of fibrils and granules. Change liver cells showed results similar to rat nephroblastoma. HeLa cells also showed rearrangement of nucleolar components after smaller drug dosage.

The results indicate that early after 4-NQO exposure nucleoli show loss of RNA with rearrangement of other components. Nuclear inclusions observed at 18–24 hr after treatment consist at least partly of nonhistone protein similar to that in untreated nucleoli. The ultrastructural findings suggest that this protein is in various states of aggregation and that unidentified elements also are present. Although the nuclear inclusions are thought to represent 4-NQO-altered residua of nucleoli, the possibility exists that they are newly formed aberrant structures.

¹ Supported by grants from American Cancer Society, E342, NIH Ca07492, and Jennie Levine League.

Received 12/17/65. Accepted 5/17/66.