Adriamycin-induced neoplastic lesions in the Egyptian toad Bufo regularis

Injecting adult toads (Bufo regularis) subcutaneously in the dorsal lymph sac with adriamycin at a dose of 2 mg/kg body weight, once every 3 weeks for 20 weeks, induced hepatocellular carcinomas in 20 out of 100 animals. Metastases of the primary liver tumors appeared in the kidneys of 7 toads.     

Induction of neoplasms in the Egyptian toad Bufo regularis by gibberellin A3

Force feeding the Egyptian toads (Bufo regularis) with gibberellin A3 (10 ppm) twice a week for 5 months induced neoplasms in 8 out of 50 (16%) experimental animals. Primary tumours developed in the liver (hepatocellular carcinomas). Two secondary tumours in the kidneys and another 2 in the ovaries of toads developed due to metastases from the hepatocellular carcinomas. The results show that gibberellic acid (gibberellin A3) has a carcinogenic effect in the Egyptian toads.     

Induction of neoplasms in Egyptian toads Bufo regularis by oil of chenopodium.

Feeding the Egyptian toad Bufo regularis with oil of the chenopodium plant induced hepatocellular carcinomas in 23% of the animals, and metastases of the primary liver tumors appeared in the kidneys of 6 toads. The earliest evidence of tumors appeared after 3 months of treatment. The average latent period of tumor induction was 3.6 +/- 0.4 months. It is speculated that oil of chenopodium may be one of the constituents of Chenopodium ambrosoides which is responsible for tumor induction in the toads B. regularis.     

Effect of cholic acid on tumor in the Egyptian toad

Egyptian toads, Bufo regularis, were fed with cholic acid (sodium salt) 3 times/week for 12 weeks at different dose levels (2.5, 5, and 10 mg/toad). Results obtained showed only 1 case in both the 5 and 10 mg/toad doses that gave a tumor. Toads receiving N-methyl-N-nitrosourea (MNU) 1 mg/toad, 3 times/week for 12 weeks had 36% ileum tumors (18 toads out of 50, without mortality). On the other hand, a higher dose of MNU (5 mg/toad) caused 50% mortality in the experimental animals. Toads treated with MNU at a dose level of 1 mg/toad were subjected to CA at dose levels of 2.5, 5, 10 mg/toad. They showed a 48, 66 and 76% higher incidence of ileum tumors at the three different dose levels, respectively. It is concluded that cholic acid has a promoting effect on ileum tumor evoked by MNU in toads as in mammals.     

Carcinogenicity testing of black pepper (Piper nigrum) using the Egyptian toad (Bufo regularis) as a quick biological test animal

Milled black pepper (Piper nigrum) force-fed to Egyptian toads as a suspension in amphibian saline or injected subcutaneously in the dorsal lymph sac as an ethanol extract, induced primary tumours in the liver and secondary tumours in other organs (kidney and spleen). When applied to the skin of experimental animals as an ethanol extract, black pepper induced primary tumours in the liver and secondary tumours in the ileum and stomach. Tumours of the liver were diagnosed as hepatocellular carcinomas and those of the other organs as metastases of the primary liver tumours. It is speculated that one or more constituents of black pepper may be responsible for tumour induction in the organs of the Egyptian toad Bufo regularis.     

Carcinogenic effect of biscuits made of flour infested with Tribolium castaneum in Bufo regularis

Biscuits made of flour infested with Tribolium castaneum induced the formation of hepatocellular carcinomas (22%) when force-fed to toads (Bufo regularis) at a dose level of 200 mg/50 g 3 times a week. Maximal time of exposure and observation was 16 weeks. Some metastatic deposits from the primary liver tumours were found in the kidneys. Thus, the carcinogenic potency of the biscuits was almost the same as that of the flour previously reported by the authors indicating that the temperature of the oven in which the biscuits were baked did not alter the carcinogenicity of the infested flour.     

Pathological-changes of the blood-cells in griseofulvin treated toads

Griseofulvin is a valuable oral antifungal drug, used extensively in the treatment of superficial fungal infections of man and animals. Electron microscopical studies of peripheral blood of toads force-fed with griseofulvin revealed pronounced alterations of the blood cells more or less similar to the criteria of monocytic leukaemia and were all comparable to those observed after the administration of the carcinogenic chemical 7,12 dimethylbenz(a)anthracene.     

Polycystic kidney and renal cell carcinoma in Japanese and Chinese toad hybrids

Frequent development of renal cell carcinomas in hybrids between Japanese toads (Bufo japonicus) and imported Chinese toads (Bufo raddei) was first reported by 2 of our authors in 1987. Such renal tumors of toads had never been observed previously in the laboratory. To confirm the observation and to establish a new animal model system, hybrids between female Japanese and male Chinese toads were newly generated from 3 pairs of parents and pathological changes in their kidneys were examined sequentially over 6 years. In hybrids from 2 of the 3 pairs, bilateral polycystic kidney developed at a high frequency from 3 months after fertilization, this being associated with the emergence of atypical, premalignant-appearing cells in proximal tubules. Papillary lesions developed after 12 months and renal cell carcinomas after 48 months. Such pathological changes were never seen in non-hybrid Chinese or Japanese toads. Electron microscopy showed no evidence of any viral participation. This unique toad model may prove useful for investigation of the underlying mechanisms of genetically determined renal cell carcinogenesis.     

Carcinogenic effect of force-feeding an extract of black pepper (Piper nigrum) in Egyptian toads (Bufo regularis)

50 male and 50 female Bufo regularis were treated, by force-feeding, with an extract of black pepper, at a dose level of 2 mg, 3 times a week for 5 months. The first tumors appeared after 2 months. Liver tumors (hepatocellular carcinomas, lymphosarcomas and fibrosarcomas) were found in 12 males and 18 females. Metastatic deposits of hepatocellular carcinomas were registered in the spleen, kidney, fat body and ovary.     

Rarity of microsatellite alterations in acute myeloid leukaemia.

We have analysed samples from 20 patients with acute myeloid leukaemia for microsatellite alterations by comparing constitutional DNA and DNA from leukaemic samples. Twelve microsatellites were amplified by PCR and investigated for novel bands, indicative of microsatellite instability, or for loss of heterozygosity. Out of 215 paired amplifications, no additional bands were observed at any locus in any of the samples analysed and loss of heterozygosity was found only as four loci from three patients. These results suggest that microsatellite alterations are very uncommon in acute myeloid leukaemia.     

Modulation of T leukaemic cell phenotype with phorbol ester

A panel of monoclonal antibodies and other markers (e.g., terminal deoxynucleotidyl transferase, sheep erythrocyte rosettes, peanut agglutinin) have been used in conjunction with flow cytometry and biochemical analysis to monitor the induction of maturation in human thymic (T) leukaemic cell lines by phorbol ester (TPA). Seven cell lines underwent multiple phenotypic alterations in response to TPA but were unresponsive to synthetic thymic hormones (TP5, FTS) or to other compounds (e.g. DMSO, retinoic acid) which induce maturation in other types of leukaemia. The changes parallel those observed in normal T-cell differentiation and partly reflect alterations in glycosyl transferase activity, altered synthesis of proteins and regulation of cell surface receptors (for transferrin) associated with rapid growth and metabolism. These studies further illustrate the reversibility of maturation arrest in human leukaemia and provide support for the view that leukaemia may involve regulatory defects in the coupling of proliferation and maturation. Induction or promotion of terminal differentiation in leukaemic equivalents of T-cell precursors may provide a convenient system for the study of biochemical and molecular events involved in T-cell development and diversification.     

Effectiveness of HB2 (anti-CD7)--saporin immunotoxin in an in vivo model of human T-cell leukaemia developed in severe combined immunodeficient mice.

The transplantation of the human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 into severe combined immunodeficient (SCID) mice was found to produce a disseminated pattern of leukaemia similar to that seen in man. The intravenous injection of 10(7) HSB-2 cells was associated with a universally fatal leukaemia. Histopathological examination of animals revealed the spread of leukaemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 MAb HB2 to the ribosome-inactivating protein saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immunotoxin (IT) to CD7+ HSB-2 target cells with an IC50 of 4.5 pM. When SCID mice were injected with 10(6) HSB-2 cells and then treated 8 days later with a single intravenous dose of 10 micrograms of immunotoxin there was a significant therapeutic effect evidenced by the numbers of animals surviving in the therapy group compared with untreated controls (chi 2 = 5.348, P = 0.021). These results demonstrate the useful application of human leukaemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 immunotoxin in human T-ALL.     

Relation between genetic variants of the ataxia telangiectasia-mutated (ATM) gene, drug resistance, clinical outcome and predisposition to childhood T-lineage acute lymphoblastic leukaemia.

The T-lineage phenotype in children with acute lymphoblastic leukaemia (ALL) is associated with in vitro drug resistance and a higher relapse-risk compared to a precursor B phenotype. Our study was aimed to investigate whether mutations in the ATM gene occur in childhood T-lineage acute lymphoblastic leukaemia (T-ALL) that are linked to drug resistance and clinical outcome. In all, 20 different single nucleotide substitutions were found in 16 exons of ATM in 62/103 (60%) T-ALL children and 51/99 (52%, P = 0.21) controls. Besides the well-known polymorphism D1853N, five other alterations (S707P, F858L, P1054R, L1472W, Y1475C) in the coding part of ATM were found. These five coding alterations seem to occur more frequently in T-ALL (13%) than controls (5%, P = 0.06), but did not associate with altered expression levels of ATM or in vitro resistance to daunorubicin. However, T-ALL patients carrying these five coding alterations presented with a higher white blood cell count at diagnosis (P = 0.05) and show an increased relapse-risk (5-year probability of disease-free survival (pDFS) = 48%) compared to patients with other alterations or wild-type ATM (5-year pDFS = 76%, P = 0.05). The association between five coding ATM alterations in T-ALL, their germline presence, white blood cell count and unfavourable outcome may point to a role for ATM in the development of T-ALL in these children.     

Concurrent translocation of BCL2 and MYC with a single immunoglobulin locus in high-grade B-cell lymphomas.

B-cell leukaemia or lymphoma with a combination of t(8;14)(q24;q32) of Burkitt leukaemia/lymphoma and t(14;18)(q32;q21) of follicular lymphoma may present clinically as de novo acute lymphoblastic leukaemia or transformation of follicular lymphoma to aggressive histology diffuse lymphoma. A number of cell lines have been reported with a complex t(8;14;18) with fusion of MYC, IGH and BCL2 on the same derivative 8 chromosome. The objective of this study was to determine the frequency and chromosomal features of this der(8)t(8;14;18) in a series of acute leukaemias and malignant lymphomas. A database of 1350 leukaemia and lymphoma karyotypes was searched for cases with structural alterations affecting both 8q24 and 18q21. A total of 55 cases were identified, of which eight revealed a complex der(8)t(8;14;18) with an MYC-IGH-BCL2 rearrangement resulting from translocation of BCL2 and MYC with a single disrupted IGH allele. Molecular cytogenetic investigation is essential to identify cases of high-grade leukaemia/lymphoma with concurrent translocations affecting the BCL2 and MYC loci.     

Mycotic Flora of the Intestine and other Internal Organs of Certain Reptiles and Amphibians with Special Reference to Characterization of Basidiobolus Isolates: Die Pilzflora des Darmes und anderer innerer Organe von bestimmten Reptilien und Amphibien unter besonderer Berücksichtigung der Charakterisierung von Basidiobolus-Isolaten

Summary: The occurrence of pathogenic fungi was investigated in intestines, lungs, livers and spleens of 200 lizards (Agama agama), 50 wall geckos (Hemiolactylus sp.), 20 toads (Bufo bufo) and 10 turtles (Chelonia mydas). The most important pathogenic fungus isolated was Basidiobolus haptosporus. It was recovered from intestinal contents of 125 (62.5%) lizards, 5 (10%) geckos, and a toad. Other important fungi isolated included 2 isolates of Allescheria boydii from lungs, 10 of Aspergillus fumigatus from lungs and intestines, and 5 of Geotrichum candidum from intestines of lizards. The yeast-like fungi were represented by 6 isolates of Candida parapsilosis from intestines of a turtle and 2 toads, lungs of 2 lizards and from the lungs and liver of a toad, 3 of Candida sp. and 2 of Trichosporon sp. from intestines of lizards, and 1 of Pichia burtoni from the lungs of a lizard. The liver of the toad yielding C. parapsilosis showed abscess like pustules on its surface; direct microscopic examination of a pustule in 10% KOH showed budding yeast cells but histology did not reveal any invasion of the tissue by the fungus. None of the other animals yielding fungal cultures revealed any gross or microscopic evidence of fungal infection of the organs. The significance of the findings has been discussed. Zusammenfassung: Bei 200 Eidechsen (Agama agama), 50 Mauergeckos (Hemiolactylus sp.), 20 Kröten (Bufo bufo) und 10 Schildkröten (Chelonia mydas) wurde das Vorkommen von Pilzen im Darm, in der Lunge, in der Leber und in der Milz untersucht. Als wichtigster pathogener Pilz wurde Basidiobolus haptosporus gefunden. Er wurde aus dem Darminhalt von 125 (62,5%) Eidechsen, 5 (10%) Geckos und bei einer Kröte gefunden. Andere wichtige Pilze, die gefunden wurden, waren 2 Isolate von Allescheria boydii aus der Lunge, 10 Stämme von Aspergillus fumigatus aus Lunge und Darm sowie 5 Stämme von Geotrichum candidum aus dem Darm von Eidechsen. Als hefeartige Pilze wurden 6 Isolate von Candida parapsilosis aus dem Darm einer Schildkröte und von 2 Kröten, aus der Lunge von 2 Eidechsen und aus der Lunge und Leber von einer Kröte gefunden. Ferner wurde 3 × Candida spezies und 2 × Trichosporon spezies aus dem Darm von Eidechsen sowie 1 × Pichia burtoni aus der Lunge einer Eidechse isoliert. Die Leber einer Kröte aus der Candida parapsilosis isoliert worden war, zeigte abszessartige Pusteln an der Oberfläche. Die direkte mikroskopische Untersuchung der Pusteln mit 10% KOH ließ Sproßzellen erkennen. In der Histologie fand sich jedoch keinerlei invasives Wachstum des Pilzes im Gewebe. Bei keinem der anderen Tiere, von denen Pilze isoliert worden waren, zeigten sich makroskopische oder mikroskopische Anzeichen einer mykotischen Infektion der Organe. Die Bedeutung der Befunde wird diskutiert.     

Alterations in the structural gene and the expression of p53 in rat liver tumors induced by aflatoxin B1.

Rat hepatocellular carcinomas (HCCs) induced by aflatoxin B1 (AFB) treatment were examined for changes in the p53 tumor suppressor gene and in p53 suppressor gene expression. A high proportion of HCCs (nine of 11 tumors in six of eight animals) exhibited new p53 restriction fragments, indicating genomic alterations of one of the p53 alleles. Each tumor with an altered p53 restriction-fragment pattern exhibited a new fragment in one of two size classes (3 kb or 7 kb with EcoRI digestion) that were missing portions of the 3' end of the p53 gene. These findings indicate that apparently similar genomic rearrangements or deletions occurred independently in AFB-induced tumors. When compared with nontumor liver tissue from the same animal, the tumors with p53 gene alterations showed dramatically reduced levels of p53 mRNA and protein and greatly increased levels of histone H2B and retinoblastoma tumor suppressor (Rb) mRNA. In two HCCs showing no evidence of p53 restriction-fragment alterations, mutant p53 protein was detected. Mutant protein was also detected in two liver samples containing an adenoma and altered foci. These data suggest that alterations of the p53 tumor suppressor gene are involved in the induction of rat HCC by AFB.     

A rodent model for testicular involvement in acute lymphoblastic leukaemia.

The testis is the third common site of relapse after primary treatment of childhood acute lymphoblastic leukaemia, but in adults relatively few testicular relapses of acute lymphoblastic leukaemia have been reported. In the present investigation the differences in the behaviour of leukaemia in immature and mature rat testis and the interactions of testicular and leukaemic cells were studied. Intraperitoneal injection of rat T-leukaemic cells to sexually immature animals induced testicular infiltrations in 100% of animals in 17 days. The infiltrations were small and located perivascularly in the interstitial tissue. Intraperitoneal injection of T-leukaemic cells to sexually mature animals induced testicular infiltrates in 42% of the animals. Leukaemic cells injected directly to the lymph sinusoids of sexually immature and mature testis proliferated rapidly causing testicular enlargement. The M(r) > 5 K fraction of extracts of 50 days old normal rat testes inhibited 3H-TdR incorporation of both normal and leukaemic ConA-stimulated rat lymphoblasts significantly. The same fraction of extracts of testes of 25 days old rats did not have any effect on 3H-TdR incorporation. The normally occurring pubertal increase in the lymphocyte inhibitory effect of the M(r) > 5 K fraction of testis extracts on 3H-TdR incorporation of PBL was prevented following either intraperitoneal or intratesticular injection of rat leukaemic lymphoblasts administered at the age of 25 days. The present observations suggest that physiological pubertal changes in the permeability of vascular endothelium and immunosuppressive effect of the testis may be important explanatory factors for the smaller number of testicular relapses in men compared to boys seen after treatment of ALL.     

Genetic alterations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the commonest form of leukaemia in adults in Western countries, is a genetically heterogeneous disease. The most frequent genetic alterations are deletions in 13q14, 17p13 (TP53) and 11q22–q23 (ATM), and trisomy of chromosome 12. Furthermore, additional alterations have been described. The most relevant techniques used for detection of genetic alterations in CLL include comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH). Recently, PCR-based techniques, such as multiplex ligation-dependent probe amplification (MLPA), have been used to detect genetic alterations in CLL. This review summarises the genetic alterations described in CLL and the techniques used for their detection.     

Inducers of Friend leukaemic cell differentiation in vitro--effects of in vivo administration.

Studies were conducted of the in vivo therapeutic potential of compounds which induce the differentiation of Friend leukaemia cells (FLC) in vitro. DBA2/J mice were inoculated with Friend leukaemia cells grown in tissue culture and at various times thereafter were treated with either N-methylacetamide, dimethylacetamide, or tetramethylurea. While survival was only occasionally prolonged, in every study these agents significantly inhibited leukaemia cell proliferation in the spleen and to a lesser extent in the marrow. These agents had no effect on the rate of proliferation of FLC growing subcutaneously nor on the proliferation of myeloid leukaemia in RFMS mice. These studies indicate that the administration of inducing agents to mice bearing Friend leukaemia can alter the proliferation characteristics of the leukaemia cells and hence suggest that these agents may have therapeutic potential.     

Life span, leukaemia and amyloid incidences of untreated and polycation-treated AKR mice

AKR mice, which have a short mean survival time and usually die with leukaemia, were studied from one month of age for correlation between these two parameters. For untreated animals we found the same mean survival time whether or not leukaemia occurred. By treating sucklings with the polycations diethylaminoethyl-dextran or hexadimethrine bromide the leukaemia incidence was significantly reduced. However, the mean survival time was unchanged, and remained the same in leukaemic and non-leukaemic animals. It is therefore suggested that the early death of AKR mice results from an ageing process and does not require leukaemia for implementation. Our prophylactic polycation treatment was furthermore found to induce spleen amyloid in some but not all of the mice that remained non-leukaemic.