Martin Cano

An in vitro model for endothelial permeability: Assessment of monolayer integrity

SummaryAn essential component of anyin vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9±11.4 Ω · cm2, 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to3H-inulin demonstrated a linear relationship between the luminal concentration of3H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of3H-inulin, was 2.01±0.076 × 10−4 ml/min (r2=.9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17±0.427×10−6 cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07±0.617×10−6 cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to3H-inulin (1.43±0.445×10−5 cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.

Removal of toxic metals and nonmetals from contaminated water

The effects of the application of potassium ferrate to remove possible toxic compounds are presented. Potassium ferrate (K2FeO4) is shown in this work to be an effective means to remove toxic metals and nonmetals from aqueous solution. The toxic material present in water is precipitated from aqueous solution and readily removed. Potassium ferrate removes itself from solution. Discolored contaminated water may be made clear by utilizing potassium ferrate. In addition, turbidities of solutions induced by dissolved substances are eliminated by the action of potassium ferrate. The efficacy of potassium ferrate in cleaning contaminated water shows great potential in application to municipal and industrial waste water.

Extensive Handling of Rats Leads to Mild Urinary Bladder Hyperplasia

The urinary and urothelial effects of the frequent handling necessary for obtaining fresh-voided urine specimens were evaluated in 5-wk-old male F-344 rats fed control diet or diet containing 7.5% sodium saccharin. Frequent handling consisted of holding rats by the back of the neck in a position to obtain fresh-voided urine directly into centrifuge tubes 3 times per week for 10 weeks, whereas seldomly handled control rats received this treatment only twice during the entire 10 weeks. The urothelium of frequently handled rats fed control diet showed superficial necrosis and regenerative hyperplasia as observed by light and scanning electron microscopy. These changes were not observed in rats fed control diet that were seldomly handled. The necrosis and hyperplasia were not as pronounced in frequently handled rats fed control diet as in seldomly handled, sodium-saccharin-treated rats, but handling also potentiated the severity of the changes produced by sodium saccharin feeding. The urothelial exfoliation and consequent regenerative hyperplasia are likely secondary effects of stress.

A comparison of the effects of sodium saccharin in NBR rats and in intact and castrated male F344 rats.

High doses of sodium saccharin (NaSac) increase proliferation in the bladder of the rat, with a male preponderance. The possibility that alpha 2u-globulin is involved in its mechanism of action was evaluated by feeding it at 7.5% of the diet to NCI-Black-Reiter (NBR) male rats, which do not synthesize liver-derived alpha 2u-globulin. NaSac affected urinary parameters similarly in F344 and NBR male rats, but NBR rats consumed more water leading to greater urinary volume. NaSac produced less proliferation in NBR than in intact F344 rats, with intermediate changes in castrated F344 males, which had intermediate urinary alpha 2u-globulin levels.

ESTABLISHMENT AND CHARACTERIZATION OF A NEW, SPONTANEOUSLY IMMORTALIZED, PANCREATIC DUCTAL CELL LINE FROM THE SYRIAN GOLDEN HAMSTER

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF-α and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-α, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.

IN VITRO PANCREATIC DUCTAL CELL CARCINOGENESIS

Our experiments were designed to identify initial biochemical and biological changes that occur during pancreatic carcinogenesis. TAKA‐1, an immortal hamster pancreatic ductal cell line, was treated in vitro for up to 11 weeks with the pancreatic carcinogen N‐nitorosobis(2‐oxopropyl)amine (BOP). These treated cells were designated TAKA‐1 + BOP. The growth of TAKA‐1 and TAKA‐1 + BOP cell lines was investigated in soft agar and in hamsters intradermally. The resulting tumor from TAKA‐1 + BOP was re‐cultured in vitro and designated TAKA‐1 + BOP‐T. Mutation of c‐K‐ras and p53 oncogenes, chromosomal changes, expression of transforming growth factor alpha (TGF‐α) and epidermal growth factor (EGF) receptor and several biochemical markers were examined in all cell lines. TAKA‐1 + BOP but not TAKA‐1 cells grew in soft agar and produced an invasive tumor in vivo. However, there were no differences in cell growth rate, DNA flow cytometry, or immunohistochemical findings between the non‐transformed and transformed cells. TAKA‐1, TAKA‐1 + BOP and TAKA‐1 + BOP‐T cells all expressed mRNA of TGF‐α and EGF receptor in a comparable pattern. DNA sequence analysis following polymerase chain reaction showed that neither TAKA‐1 nor TAKA‐1 + BOP cells has a mutation of c‐K‐ras or p53. Karyotype analysis demonstrated that TAKA‐1 + BOP cells had more chromosomal abnormalities compared with TAKA‐1 cells. Mutation of c‐K‐ras and p53 was not essential for carcinogenesis in hamster pancreatic ductal cells in vitro. In conclusion, immortality of the TAKA‐1 cells caused expression of TGF‐α to the same extent as in malignant cells. Chromosomal and ultrastructural patterns were the only differences detected between the non‐transformed and BOP‐transformed cells. Int. J. Cancer 72:1095–1103, 1997.

Comparative studies on expression of tumor-associated antigens in human and induced pancreatic cancer in syrian hamsters

SummaryThe expression of blood-group-related antigens (BGRAs) in experimental primary pancreatic cancer induced byN-nitrosobis(2-oxopropyl)amine (BOP) treatment of Syrian hamsters and homologous subcutaneous transplants of this primary cancer in the cell line, PC-1, established from the primary cancer and intrapancreatic transplanted PC-1 cells were studied by histochemical and biochemical methods. Human primary pancreatic cancer; the human pancreatic cancer cell line, HPAF; and its subclones, CDU and CD18, also were studied on a comparative basis.Histochemical analysis of BGRAs demonstrated that A, B, H, Leb, Lex Ley, and T antigen were expressed both in vivo and in vitro in hamster and human materials in similar patterns. However, Lea, CA 19–9 and sialylated Tn antigens were not found in hamster-derived tissues. SDS-PAGE and Western blotting procedures using anti-A antigen revealed similar major bands in the membrane fractions of both human and hamster pancreatic cells between 97 and 200 kdalton. Among other human pancreatic cancerassociated antigens, TAG-72, CA 125, and 17–1A were detected immunohistochemically in the hamster tumors both in vivo and in vitro, in a pattern similar to that seen in human pancreatic cancer. Tumor antigen DU-PAN-2, associated with human pancreatic cancer, was found infrequently in hamster pancreatic cancer specimens. These results indicate that the experimental hamster pancreatic cancer model provides a unique tool for investigating antigenicity of pancreatic cancer, particularly in relation to diagnosis and therapy.

Bladder freeze ulceration and sodium saccharin feeding in the rat: Examination for urinary nitrosamines, mutagens and bacteria, and effects on hepatic microsomal enzymes

We previously demonstrated that long-term feeding of sodium saccharin, a non-mutagen, induced bladder carcinomas when administered to F344 male rats with regenerative hyperplasia of the urothelium induced by the freeze-ulceration technique, even without prior chemical initiation (Cohen et al. Cancer Res. 1982, 42, 65). In the present study, we examined the urine of rats subjected to freeze ulceration of the bladder and then fed sodium saccharin at 5% in the diet to evaluate the possibility of a mutagen being generated as a result of ulceration and/or saccharin feeding. Urine was collected into a syringe by aspiration from the urinary bladder after ligating the urethra for 2 hr at intervals from day 0 to day 14 after ulceration. After ulceration and/or sodium saccharin feeding, the urine showed no bacterial contamination, no mutagenic activity in the standard Ames assay, no production of nitrosamines, and no nitrosating environment. In addition, no significant changes in activities of liver microsomal enzymes (i.e. cytochrome P-450, NADPH-cytochrome c reductase, aniline hydroxylase, or ethylmorphine N-demethylase) were observed in rats fed sodium saccharin for 1, 5 or 14 days. Thus, freeze ulceration, and the consequent regenerative hyperplasia of the epithelium, compared with sodium saccharin feeding do not involve the administration of an exogenous mutagenic substance or the generation of a detectable mutagen in the urine.

Evaluation of diet and dimethylarsinic acid on the urothelium of Syrian golden hamsters.

Few studies have examined the carcinogenicity of chemicals toward the urinary bladder in hamsters, and the effect of diet on hamster urine and urothelium has not been reported. Our laboratory recently began investigating the effects of dimethylarsini c acid (DMA) on the hamster bladder, and we noticed subtle urothelial changes even in controls. The possible effect of various diets on hamster urothelium was evaluated by feeding different diets to 4-week-old Syrian Golden hamsters for 5 weeks. The diets examined were Tekland 8656, Purina 5002, Purina 5L79, and NIH-07. Light microscopic examination showed a slight increase in urothelial hyperplasi a in hamsters fed Purina 5L79. An increase in the incidence of urinary bladder necrosis, exfoliation, and mild hyperplasi a were noted by scanning electron microscopy (SEM) with all dietary preparation s except NIH-07. The constituents in the diets producing the urothelial alterations are not known at present, but NIH-07 diet was chosen for experiments to investigate the effects of DMA on the hamster bladder epithelium. Male and female 5-week-old Syrian Golden hamsters were fed 100 ppm DMA for 10 weeks. Examination of urinary parameters showed no treatment-related changes. Light microscopic examination and SEM revealed no changes of the urothelium of DMA-treated male or female hamsters.

Morphological lesions of the rat urinary tract induced by inoculation of mycoplasmas and other urinary tract pathogens

The effects on the urinary tract after inoculation of Ureaplasma urealyticum into the rat bladder were evaluated and compared to that seen after Mycoplasma hominis, Escherichia coli and Proteus mirabilis inoculation. The inoculation of the urease-producing organisms P. mirabilis and U. urealyticum were associated with the formation of struvite bladder stones and predominantly hyperplastic lesions of the bladder. The P. mirabilis inoculated rats also displayed marked pyelonephritis. A similar but much less pronounced reaction also occurred in the kidneys of some of the U. urealyticum inoculated rats. P. mirabilis could frequently be recultured. In contrast, this was not possible with U. urealyticum, but the organism was detected by scanning electron microscopy 2 weeks after the inoculation. Inoculation of M. hominis was associated with a few mild lesions of the bladder, but inflammatory lesions were not present in the kidneys. The study confirms the potential of Ureaplasma to form struvite stones in rat urinary tract. It also demonstrates that it can induce inflammatory changes in both bladder and kidney of rats without concomitant stone formation.