Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of moxifloxacin, a quinolone antibiotic

The chronic toxicity and carcinogenicity of Moxifloxacin (MOX), a bacterial gyrase-inhibiting fluoroquinolone antibiotic, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity study designed to assess potential carcinogenic activity of a test substance in critical organs in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder, mammary gland, bone marrow, thymus, spleen and stomach. MOX was given daily by intragastric instillation at 500 mg/kg bw/day for the first 13 weeks to produce potential initiation, followed by promoters (PROs) for 24 weeks, or for the last 24 weeks after 13 weeks of exposure to initiators (INs). The INs, administered during the first 13 weeks, were diethylnitrosamine for the liver, N-n-butyl-N-(4-hydroxybutyl)nitrosamine for the urinary bladder, ethylnitrosourea for the hematolymphoreticular system, N-nitrosodimethylamine for lungs, methylnitrosourea for the stomach and 7,12-dimethylbenz(a)-anthracene for the mammary gland. The PROs, administered during the last 24 weeks after MOX, were phenobarbital for the liver, nitrilotriacetic acid for the urinary bladder, azathioprine for the bone marrow, butylated hydroxytoluene for the lung, butylated hydroxyanisole for the forestomach, and diethylstilbestrol for the mammary gland. The INs produced preneoplastic and neoplastic lesions which were not enhanced by MOX, and MOX plus PROs elicited no neoplastic effects, documenting that MOX did not produce either initiation or promotion of neoplasia in any of the target sites, or in any of the other twenty tissues examined.     

Modifying effects of antioxidants on chemical carcinogenesis

Studies were made on the carcinogenic activity of butylated hydroxyanisole (BHA) in rats, mice, and hamsters and the effect of the antioxidants BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), sodium L-ascorbate (SA), ascorbic acid (AA), sodium erythorbate (SE), propyl gallate (PG), and alpha-tocopherol, on two-stage chemical carcinogenesis in rats initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 1,2-dimethylhydrazine (DMH), diethylnitrosamine (DEN), 7,12-dimethylbenz(a)anthracene (DMBA), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-ethyl-N-hydroxyethylnitrosamine (EHEN), or N-methylnitrosourea (MNU). BHA clearly induced squamous cell carcinomas in both the rat and hamster forestomach. The tumorigenic action of crude BHA on the forestomach is largely due to 3-tert-BHA. In two-stage chemical carcinogenesis, BHA promoted MNNG or MNU-initiated forestomach and BBN- or MNU-initiated urinary bladder carcinogenesis and inhibited DEN- or EHEN-initiated liver and DMBA-initiated mammary carcinogenesis. BHT demonstrated promotion potential for urinary bladder and MNU-initiated thyroid carcinogenesis and inhibited DMBA-initiated ear duct carcinogenesis. EQ promoted EHEN-initiated kidney carcinogenesis and inhibited DMBA-initiated mammary and EHEN-initiated liver carcinogenesis. SA promoted forestomach and urinary bladder carcinogenesis and SE likewise enhanced urinary bladder carcinogenesis. alpha-Tocopherol inhibited ear duct carcinogenesis. No effects of any of the antioxidants on glandular stomach carcinogenesis were found. The results clearly demonstrated that antioxidants have different effects (promoting or inhibitory influences) depending on the organ studied and suggest the importance of a whole body approach to their investigation.     

The histopathological and biochemical response of the stomach of male F344/N rats following two weeks of oral dosing with ethyl acrylate

Male F344/N rats were dosed with ethyl acrylate (EA) either by daily gavage or in the drinking water for 2 weeks. The gavage dose levels were 0, 2, 10, 20, 50, 100, and 200 mg/kg; the drinking water dose concentrations were 0, 200, 1,000, 2,000, and 4,000 ppm (corresponding to 0, 23, 99, 197, and 369 mg/kg/day, respectively). In those animals dosed by gavage, irritation of the forestomach increased in incidence and severity over the 20-200 mg/kg dose range. In those animals dosed with EA in the drinking water, a much lower incidence of forestomach irritation and less severe lesions were observed at corresponding dose levels. No lesions were observed in the glandular stomach from either of the 2 modes of oral administration. Following 2 weeks of gavage dosing with EA, the total non-protein sulfhydryl (NPSH) content of the forestomach and glandular stomach, and the NPSH concentration of the liver were determined 2-24 hr after the last gavage dose. Animals dosed at 200 mg/kg reached approximately 11% of the initial NPSH content in the forestomach at 6 hr after dosing. NPSH depletion of this magnitude has been associated with cytotoxicity of other tissues in other studies. By contrast, either the glandular stomach nor liver were depleted of NPSH to levels generally associated with toxicity. These observations are consistent with the conclusion that bolus dosing of EA induces severe depletion of critical cellular thiols in the forestomach with toxic consequences, but not in the glandular stomach or liver. Changing the mode of oral administration for EA to continued small doses in the drinking water allowed efficient detoxification and did not induce sulfhydryl depletion or comparable forestomach toxicity at the same daily body burden.     

Inhibitory effect of peptide vilon on the development of induced rat urinary bladder tumors in rats

The effect of peptide vilon (Lys-Glu) on urinary bladder carcinogenesis in rats was studied. Urinary bladder tumors were induced with a selective carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine. The tumors developed in 56% vilon-treated animals and in 75.5% controls. Vilon 2-fold decreased the incidence of preneoplastic and early neoplastic changes in urinary bladder mucosa and significantly inhibited carcinogenesis.     

Dietary restriction alters cell proliferation in rats: an immunohistochemical study by labeling proliferating cell nuclear antigen.

Dietary restriction (DR) delays the onset of aging and lowers the incidence of both spontaneous and chemically induced cancers. The inhibition of cell proliferation has been suggested as a possible mechanism for this effect. We examined the effect of DR on cell proliferation in duodenum, forestomach, glandular stomach, and liver tissues of male Fischer 344 rats receiving 60% of the control feed intake for 24 months starting at 16 weeks of age. Rats were sacrificed, when 28 months old. Tissues were collected, histologically prepared, and stained immunohistochemically for proliferating cell nuclear antigen (PCNA). The PCNA-stained nuclei are detected in different phases of the cell cycle. A minimum sample of 2000 cells was counted in liver. The percentage of labeled S-phase cells per total cells counted was used as the labeling index for liver. The number of labeled S-phase epithelial cells per 1.1 mm of basement membrane or muscularis mucosa was used as the labeling index for duodenum, forestomach, and glandular stomach. Cell proliferation in glandular stomach and liver tissues was inhibited in rats DR for 24 months; however, cell proliferation in duodenum and forestomach mucosal tissues was unexpectedly enhanced by DR. These results indicated that while DR inhibits cell proliferation in tissues of rats, it is tissue-dependent. The decreased rate of cell division by DR in the designated tissues could be implicated in lowering the conversion of endogenous DNA damage or lesions to mutation and cancer.     

Incidence, distribution, and morphology of amyloidosis in Charles Rivers CD-1 mice.

The incidence, morphology, and distribution of amyloidosis were reviewed in a 2-year toxicity-oncogenicity study in Charles Rivers CD-1 mice. Amyloid was present in the duodenum, jejunum, mesenteric lymph node, and ovary in animals sacrificed at 8 months. In animals sacrificed at 12 months, amyloid was also present in the adrenal gland, gall bladder, heart, ileum, kidney, pancreas, parathyroid, spleen, glandular stomach, testis, and thyroid. In the animals sacrificed at 24 months, the gland was also involved. The organs most frequently involved at 24 months included the adrenal gland, duodenum, jejunum, ileum, heart, kidney, liver, mesenteric lymph node, ovary, spleen, and thyroid.     

Cytogenetic abnormalities in MALT lymphomas and their precursor lesions from different organs. A fluorescence in situ hybridization (FISH) study

Aims: To analyse the possible activation of distinct molecular pathways in mucosa‐associated lymphoid tissue (MALT) lymphoma, we determined the prevalence of trisomies 3, 12, 18 in MALT lymphomas from different organs, as well as the prevalence of translocations of the MALT1 gene in a subset of primary breast MALT lymphomas. We compared the numerical cytogenetic alterations in lymphomas, precursor lesions and in normal non‐haematolymphoid tissue from the same organs. Methods and results: Forty‐two samples of paraffin‐embedded tissue (29 MALT lymphomas from stomach, breast, parotid and thyroid; two Sjögren's syndrome; two Hashimoto's thyroiditis and nine reactive samples) were studied by fluorescence in situ hybridization (FISH). Analysed together, the cases of gastric, parotid and thyroid MALT lymphomas presented trisomy 3 in 46%, trisomy 12 in 28% and trisomy 18 in 21% of the cases. In contrast to other locations, trisomy 3 was not present in the majority of the cases of primary breast MALT lymphomas. None of the nine breast cases presented MALT1 gene rearrangements. Half of the cases of preneoplastic lesions exhibited trisomy 3 and trisomy 12; none exhibited trisomy 18. Conclusions: Trisomy 3 is the most frequent numerical abnormality in gastric, parotid and thyroid but not in primary breast MALT lymphomas. MALT1 gene rearrangements are also rare in this location, suggesting that distinct molecular pathways may be activated in breast cases.     

Hyalinosis and Ym1/Ym2 gene expression in the stomach and respiratory tract of 129S4/SvJae and wild-type and CYP1A2-null B6, 129 mice

The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. The possible cause of this protein excess in gastric and other lesions and its possible functions are discussed.     

Carcinogenic susceptibility to N-bis(2-hydroxypropyl)nitrosamine (DHPN) in rasH2 mice

To evaluate the susceptibility of rasH2 mice to N-bis(2-hydroxypropyl)nitrosamine (DHPN), a potent carcinogen targeting the lung, liver, thyroid, and kidney, male, 6-week old, rasH2 mice and wild-type littermates (non-Tg mice) were given DHPN in drinking water at 0, 20 or 200 ppm, and 0 or 200 ppm, respectively, for 26 weeks. The experiment using rasH2 mice given 200 ppm DHPN and non-Tg mice given 200 and 0 ppm DHPN was completed at 20 weeks, since mortality in these groups was remarkably increased due to hemangiosarcomas of the liver. Histologically, tumors developed in the lung and liver in both rasH2 and non-Tg mice treated with DHPN. In addition, proliferative lesions were observed in the forestomach, urethra, and excretory duct of salivary glands in rasH2 mice given 200 ppm DHPN. RT-PCR analysis showed no marked difference in expression of mRNAs for the transgene and the endogenous mouse ras gene between the whole lung tissue containing a neoplasm and normal lung tissue. Our results suggest that rasH2 mice are highly susceptible to DHPN, the target organs including the forestomach, salivary gland and urethra, which have not been found to develop tumors in previous long-term carcinogenicity studies of DHPN in rats and mice.     

Glandular lesions of the urinary bladder:clinical significance and differential diagnosis

Williamson S R, Lopez‐Beltran A, Montironi R & Cheng L
(2011) Histopathology  58 , 811–834
Glandular lesions of the urinary bladder: clinical significance and differential diagnosis A variety of glandular or pseudoglandular lesions may be seen in the urinary bladder, ranging from those that are entirely benign to aggressive‐behaving malignant primary and secondary tumours. Lesions with minimal to no evident premalignant potential include several proliferative and reactive processes, such as cystitis cystica and cystitis glandularis, although the possibility exists for confusion of such lesions with an infiltrative neoplasm, particularly in limited biopsy specimens. Similarly, ectopic tissues of Müllerian origin may be seen occasionally in the urinary bladder and their differentiation from a true glandular neoplasm is important to avoid improper treatment. As urothelial carcinoma has a propensity for divergent differentiation, a wide spectrum of morphological variants exists with varying degrees of glandular differentiation. Some such variants have demonstrated clinical behaviour that is more aggressive than their histology would suggest, thus deserving recognition and potentially different treatment. In this paper, we review the glandular lesions of the urinary bladder ranging from benign proliferative processes to malignant primary and secondary neoplasms, with emphasis on clinical significance and features useful in resolving their differential diagnoses.     

Loss of GATA4 causes ectopic pancreas in the stomach

Pancreatic heterotopia is defined as pancreatic tissue outside its normal location in the body and anatomically separated from the pancreas. In this work we have analyzed the stomach glandular epithelium of Gata4 ^flox/flox; Pdx1-Cre mice (Gata4KO mice). We found that Gata4KO glandular epithelium displays an atypical morphology similar to the cornified squamous epithelium and exhibits upregulation of forestomach markers. The developing gastric units fail to form properly, and the glandular epithelial cells do not express markers of gastric gland in the absence of GATA4. Of interest, the developing glands of the Gata4KO stomach express pancreatic cell markers. Furthermore, a mass of pancreatic tissue located in the subserosa of the Gata4KO stomach is observed at adult stages. Heterotopic pancreas found in Gata4-deficient mice contains all three pancreatic cell lineages: ductal, acinar, and endocrine. Moreover, Gata4 expression is downregulated in ectopic pancreatic tissue of some human biopsy samples. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The degradation of histamine in some animal species

The aim of this work was 1. determine the major [³H]-metabolites of histaminein vivo by using the musculus gracilis of the dog, andin vitro in different tissues of the cat, 2. determine the endogenous levels of histamine and N^τ-methylhistamine in some tissues of the cat and the rat.

1. [³H] histamine injection into arterial supply of the muscle of the dog: after 60 minutes the content of [³H] histamine in the muscle was 1.9\+-0.6% and the content of [³H] N^\gt-methylhistamine and [³H] \lsAcid metabolites\rs was 47\+-6.7% and 51.0\+-6.5%. The cat tissuesin vitro showed the following t_1/2 for exogenous histamine: submandibular gland, 95.9 min, parotid gland, 36.1 min, stomach pylorus, 39.5 min, thyroid gland, 28.3 min, liver, 23.7 min.
2. Endogenous levels of histamine and N^\gt-methylhistamine were determined in the cat and the rat. In general, the contents of endogenous histamine and N^\gt-methylhistamine were similar to the data obtained with labelled [³H] histamine. In both species submandibular gland was among the richest tissues in N^\gt-methylhistamine content. The provenience of N^τ-methylhistamine in mast cells is discussed.

     

Pyloric gland metaplasia/differentiation in multiple organ systems in a patient with Peutz-Jegher's syndrome

Peutz-Jegher's syndrome (PJS) involves multiple organ systems and the development of hamartomatous, metaplastic, or neoplastic lesions of different cell lineages. Among them, glandular lesions are the most common, but their properties are obscure. We report here a 53-year-old woman with PJS who developed multiple hamartomatous polyps in the jejunum and mucinous glandular lesions in multiple organ systems: glandular metaplasia in the urinary bladder; lobular endocervical glandular hyperplasia in the uterine cervix; mucinous metaplasia in the right fallopian tube; mucinous adenoma in the left ovary. Histological and immunohistochemical analyses disclosed that all of the intestinal and extra-intestinal lesions were associated with pyloric gland metaplasia/differentiation across the organ systems. In the general population, the organs described above rarely or infrequently show pyloric gland phenotype, to say nothing of trans-organ involvement. It is strongly suggested that commitment to pyloric gland metaplasia/differentiation is closely associated with PJS.     

ACB‐PCR measurement of H‐ras codon 61 CAA→CTA mutation provides an early indication of aristolochic acid I carcinogenic effect in tumor target tissues

Aristolochic acid (AA) is a strong cytotoxic nephrotoxin and carcinogen, which induces forestomach and kidney tumors in mice and is associated with development of urothelial cancer in humans. This study sought to gain mechanistic insight into AAI‐induced carcinogenesis through analysis of a tumor‐relevant endpoint. Female Hupki mice were treated daily with 5 mg AAI/kg body weight by gavage for 3, 12, or 21 days. Histopathology and DNA adduct analysis confirmed kidney and forestomach as target tissues for AAI‐induced toxicity. H‐ras codon 61 CAA→CTA mutations were measured in mouse kidney and forestomach, as well as liver and glandular stomach (nontarget organs) by allele‐specific competitive blocker‐PCR (ACB‐PCR), because A→T transversion is the predominant mutation induced by AA and this particular mutation was found previously in AA‐induced rodent forestomach tumors. Treatment‐related differences were observed, with the H‐ras mutant fraction (MF) of mouse kidney and forestomach exposed to 5 mg AAI/kg body weight for 21 days significantly higher than that of vehicle‐treated controls (Fisher's exact test, P < 0.05). Statistically significant correlations between dA‐AAI adduct levels (measured previously in the same animals) and induced H‐ras MFs were evident in forestomach of mice treated for 21 days (linear regression, P < 0.05). The significant increase in H‐ras MF in kidney and forestomach, along with the correlation between DNA adducts, histopathology, and oncogene mutation, provide definitive evidence that AA induces tumors through a directly mutagenic mode of action. Thus, measurement of tumor‐associated mutations is a useful tool for elucidating the mechanisms underlying the tissuespecificity of carcinogenesis. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Toxicity, DNA binding, and cell proliferation in male F344 rats following short-term gavage exposures to trans-2-hexenal

Hexenal is a genotoxic compound to which humans are exposed daily through the consumption of foods and beverages. The present studies were conducted to examine the relationships between the dose-responses of trans-2-hexenal-induced toxicity, DNA adduct formation, and cell proliferation. Male F344 rats were exposed by gavage to single doses of up to 500 mg/kg and killed 1, 2, or 4 days after dosing or were exposed to repeat doses of up to 100 mg/kg once daily for 5 days or 5 days per week for 4 weeks and killed 1 day after the end of the dosing period. Histologically, the primary observations were necroulcerative lesions, inflammation, and hyperplasia in the forestomach and inflammation in the glandular stomach. Hexenal-derived DNA adduct formation and cell proliferation were induced in the forestomach at doses of hexenal that also induced gastric toxicity; DNA adducts were not observed in the glandular stomach. These findings suggest that the toxicity of hexenal was limited to the site of contact (stomach) and that the observed DNA adduct formation and cell proliferation occurred in the setting of severe tissue damage.     

Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis

Cytokeratins (CKs) 14 and 20 are promising markers for diagnosing urothelial lesions and for studying their prognosis and histogenesis. This work aimed to study the immunohistochemical staining patterns of CK14/20 during multistep carcinogenesis leading to papillary bladder cancer in a rat model. Thirty female Fischer 344 rats were divided into three groups: group 1 (control); group 2, which received N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) for 20 weeks plus 1 week without treatment; and group 3, which received BBN for 20 weeks plus 8 weeks without treatment. Bladder lesions were classified histologically. CK14 and CK20 immunostaining was assessed according to its distribution and intensity. In control animals, 0–25% of basal cells and umbrella cells stained positive for CK14 and CK20 respectively. On groups 2 and 3, nodular hyperplastic lesions showed normal CK20 and moderately increased CK14 staining (26–50% of cells). Dysplasia, squamous metaplasia, papilloma, papillary tumours of low malignant potential and low‐ and high‐grade papillary carcinomas showed increased CK14 and CK20 immunostaining in all epithelial layers. Altered CK14 and CK20 expression is an early event in urothelial carcinogenesis and is present in a wide spectrum of urothelial superficial neoplastic and preneoplastic lesions.     

The human papillomavirus type 16 E7 oncoprotein targets Myc-interacting zinc-finger protein-1

We demonstrate that HPV-16 E7 forms a complex with Miz-1. UV-induced expression of the CDK-inhibitor p21^Cip1 and subsequent cell cycle arrest depends upon endogenous Miz-1 in HPV-negative C33A cervical cancer cells containing mutated p53. Transient expression of E7 in C33A inhibits UV-induced expression of p21^Cip1 and overcomes Miz-1-induced G1-phase arrest. The C-terminal E7Δ79LEDLL83-mutant with reduced Miz-1-binding capacity was impaired in its capability to repress p21^Cip1 expression; whereas the pRB-binding-deficient E7C24G-mutant inhibited p21^Cip1 expression similar to wild-type E7. Using ChIP, we demonstrate that endogenous E7 is bound to the endogenous p21^Cip1 core-promoter in CaSki cells and RNAi-mediated knock down of Miz-1 abrogates E7-binding to the p21^Cip1 promoter. Co-expression of E7 with Miz-1 inhibited Miz-1-induced p21^Cip1 expression from the minimal-promoter via Miz-1 DNA-binding sites. Co-expression of E7Δ79LEDLL83 did not inhibit Miz-1-induced p21^Cip1 expression. E7C24G retained E7-wild-type capability to inhibit Miz-1-dependent transactivation. These findings suggest that HPV-16 E7 can repress Miz-1-induced p21^Cip1 gene expression.     

Fasciola hepatica human infection

Summary Sixteen human cases of Fasciola hepatica infection are described. The liver was involved in 13 cases, the gall bladder in 9 cases and the stomach in 2 cases. Lesions containing parasitic remnants or fluke eggs were rarely seen. Surface scarring of the liver, scar tracks and granulomas within organs were the most characteristic changes seen and were the most useful for the histopathological diagnosis of the disease. The associated liver, bile and gastric lesions are briefly discussed.     

A stereological study of the volume-weighted volume and of the relative volume of the nucleus of normal and preneoplastic hepatocytes in a trout model of hepatocarcinogenesis

The fish liver has been the main subject of the biomonitoring and laboratory studies dealing with environmental carcinogenesis. The foci of cellular alterations are accepted pre-neoplastic hepatic lesions, and histopathology is the primary tool for their characterization. Despite its potential, using stereology to study quantitatively nuclear features of those lesions has not been evaluated. Herein, we estimated the volume density and the volume-weighted volume of the nucleus of normal and preneoplastic hepatocytes, using stereology and the brown trout (Salmo trutta f. fario) as model. In the hepatocarcinogenesis protocol the N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was used as initiator, and the 17-β estradiol (E2) as promoter. Three groups of 30 animals were considered: negative controls (non-exposed), initiator exposed and initiator plus promoter exposed. Estimates of both stereological parameters were significantly higher in preneoplastic hepatocytes, also showing an excellent discriminatory power when used to differentiate those hepatocytes from the normal ones. Besides, in the normal parenchyma the two parameters also differed among the three tested groups. The exposure to MNNG and/or to E2 leaded to modifications in the hepatocyte nuclei that could be unbiasedly quantified with two stereological parameters. We showed that quantitative nuclear morphology represents a valuable auxiliary tool in assessing hepatocarcinogenesis in fishes.     

Dose-dependent induction of preneoplastic lesions by the tobacco-specific nitrosamine carcinogen NNK in the in ovo carcinogenicity assessment (IOCA) assay

The potential of the carcinogenic tobacco specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-1-butanone (NNK) to induce preneoplastic hepatocellular altered foci (HAF) was tested in the in ovo carcinogenicity assessment (IOCA) assay. Single doses of NNK over a dose range from 0.1 mg to 6 mg were injected into fertilized turkey eggs prior to incubation for 24 days. The livers were investigated by histological, histochemical and morphometric methods. Mortality was increased for eggs exposed to 6 mg. In this group, the whole livers were severely altered, showing pronounced changes of nucleus size and signs of cell death. At the dose of 2 mg various types of foci of altered hepatocytes (HAF) were observed. Basophilic cell foci of the solid or tubular type were most frequent. The NNK-induced HAF were very similar to the preneoplastic lesions that occur in the livers of mammals during hepatocarcinogenesis which are regarded as early indicators of carcinogenesis. The similarity to the HAF in rodents included histochemically detectable alterations like decreased activities of glucose-6-phosphatase, adenosine triphosphatase and glycogen phosphorylase. At doses of 1 mg or below, no HAF were detected. At all dose levels an increased occurrence of enlarged hepatocytes with enlarged nuclei and prominent nucleoli (karyomegalic hepatocytes) were observed. The increase in karyomegalic hepatocytes was also statistically significant at the low dose of 0.1 mg/kg NNK but the dose–effect curve for their induction was clearly non-linear. Induction of HAF and karyomegalic hepatocytes in ovo is a simple (one dose), rapid (24 days) and inexpensive (no animal purchase or housing) experimental approach for studies on chemically induced hepatocarcinogenesis.