The role of proliferation in the origin of mutations in mammalian cells

Biliary obstruction, produced by common bile duct ligation or alpha-naphthylisothiocyanate (ANIT) treatment in rats, has been associated with the development of type I biliary epithelial cell (BEC) hyperplasia. However, the exact mechanism(s) by which bile duct obstruction lead(s) to this proliferative lesion are not clear. The present studies were designed to determine if cholestasis, in the absence of biliary obstruction, would result in type I BEC hyperplasia. Male Sprague-Dawley rats were given a single oral dose of 150 mg/kg ANIT or i.v. doses of estradiol glucuronide (E2-17G; 21 mumol/kg/h for 48 h) to produce obstructive and non-obstructive cholestasis, respectively. E2-17G treatment resulted in cholestasis that was comparable in extent and duration to that observed following ANIT treatment. E2-17G and ANIT treatments produced comparable increases in serum bile acids (55- to 60-fold) and activities of ALT (36- to 38-fold), ALP (4- to 5-fold), and 5'-nucleotidase (7- to 11-fold), respectively, compared to controls. Both ANIT and E2-17G also increased serum bilirubin concentrations. ANIT treatment resulted in significant increases in biliary glucose concentrations that were associated with BEC damage/necrosis and obstruction of the bile duct lumen. Conversely, no evidence of BEC damage was observed in E2-17G-treated rats. Nonetheless, BEC hyperplasia was observed in the majority of rats following treatment with either ANIT or E2-17G, assessed by light microscopy and by BrdU immunohistochemistry. These data indicate that E2-17G treatment produces nonobstructive cholestasis and type I BEC hyperplasia, suggesting that biliary obstruction is not a prerequisite for type I BEC hyperplasia in rats. Differences in the time of onset of hyperplasia were observed: hyperplasia was noted immediately following 48 h of E2-17G-induced cholestasis but occurred several days after ANIT-induced cholestasis had subsided. Since the magnitude/duration of cholestasis was similar in the two models but the temporal association between cholestasis and type I BEC hyperplasia were different, these data suggest that the proliferative stimulus may be different in the two models and that E2-17G-induced type I BEC hyperplasia may not be attributed solely to cholestasis.     

A microcholangiographic study of liver disease models in rats

RATIONALE AND OBJECTIVES: Rats develop hepatobiliary injury due to small bowel bacterial overgrowth (SBBO) that, at specimen, resembles cholangiography sclerosing cholangitis. To better visualize the smaller bile ducts, we used microcholangiography to determine the spectrum of biliary lesions in this and five other models of liver disease. METHODS: The models studied were as follows: (1) Surgically created jejunal, self-filling blind loops induce SBBO. (2) Intraperitoneal injection of a bacterial cell wall polymer, peptidoglycan-polysaccharide (PG-PS), causes granulomatous hepatitis. (3) Intraperitoneal injection of endotoxin (lipopolysaccharide) causes sinusoidal congestion and shock. (4) Bile duct ligation induces bile duct proliferation. (5) Alpha-naphthyl-isothiocyanate (ANIT) induces bile duct proliferation. (6) Carbon tetrachloride (CCl4) causes fibrosis and cirrhosis. Warmed barium sulfate, gelatin, and saline were injected in the extrahepatic bile duct. Liver slices (2 mm) underwent microradiographic techniques, and images were correlated with histology. RESULTS: Rats with SBBO had irregular and dilated extrahepatic bile ducts with thickened walls. Rats treated with endotoxin and CCl4 had normal microcholangiograms. Bile duct proliferation was identified following ANIT and bile duct ligation. Rats given PG-PS demonstrated irregular intrahepatic bile ducts. Microcholangiograms following SBBO and PG-PS showed similarities including focal ductal dilatation, narrowing, proliferation, and destruction. CONCLUSION: Various models of liver injury induce characteristic cholangiographic appearances. Microcholangiography is useful in examining biliary tract lesions and complements histology.     

Bile regulates the expression of major histocompatibility complex class II molecules on rat intestinal epithelium

BACKGROUND & AIMS: Major histocompatibility complex (MHC) class II molecules are expressed on intestinal epithelial cells, and the intensity of this expression is regulated. The aim of this study was to test the hypothesis that bile regulates the expression of MHC class II molecules on intestinal epithelium. METHODS: Rats were deprived of intestinal bile by external drainage for 24 or 48 hours, and their intestines were collected, sectioned, and stained with the anti-MHC class II monoclonal antibodies OX4 and OX6. For one group of rats, bile flow was deviated from its usual entry point to the ileum. RESULTS: Compared with intact animals, MHC class II expression was observed to be diminished within 24 hours and totally absent after 48 hours of bile drainage. For the group in which bile flow was deviated to the ileum, staining was only observed in the region distal to the entry point. Analysis by bioassay and enzyme-linked immunosorbent assay of bile showed the presence of tumor necrosis factor and interferon gamma, respectively. CONCLUSIONS: It is concluded that the presence of bile is required for the expression of MHC class II molecules on gut epithelium and that the cytokine components of bile may be the inducing agents.     

Photodynamic therapy on rat urinary bladder with intravesical instillation of 5-aminolevulinic acid: light diffusion and histological changes

PURPOSE: Photodynamic therapy (PDT) has the potential to treat extensive premalignant lesions and microinvasive tumors in the bladder, but its use has been hampered by the risk of detrusor muscle damage and prolonged skin photosensitivity. We have shown that the rat urothelium can be sensitized by selectively using a 10% solution of 5-aminolevulinic acid (ALA) at pH 5.5 administered intravesically. This paper evaluates the photodynamic effects on sensitized bladders. MATERIALS AND METHODS: The bladders ofs Wistar rats were instilled with ALA solutions of different concentrations at pH 5.5 and subsequently treated with laser light at 630 nm. Bladders were harvested 1 to 7 days after PDT for histological assessment. RESULTS: Under optimum conditions (10% intralipid diffusion medium, light dose 50J) uniform urothelial necrosis was seen after 1 to 2 days; it healed in 7 days without damage to the underlying muscle layer although some increase in collagen was seen in the lamina propria. Overtreatment or poor light distribution resulted in muscle necrosis and scarring. CONCLUSIONS: Selective urothelial necrosis is possible with PDT using intravesical ALA. There is now sufficient data for pilot clinical trials to start photodynamic therapy for management of superficial bladder cancer or carcinoma in situ.     

Clinical evaluation of 99mTc-pyridoxylideneglutamate for hepatobiliary scanning

Clinical evaluation of hepatobiliary scanning using 99mTc-PG was done in twenty normal volunteers and eighty-three patients with liver and biliary tract disease. Satisfactory images of the biliary tract were obtained using small dosages of this agent. In normal humans, the agent reached the liver in 5 minutes, and the common bile duct, gallbladder, and duodenum in 10 to 20 minutes. The gallbladder was not visualized when the cystic duct was obstructed in patients with acute and chronic cholecystitis. In patients with partial common bile duct obstruction, a distended duct was visualized and there was delay in transit of radioactivity into the duodenum. With complete common bile duct obstruction, no radioactivity was seen in the biliary or gastrointestinal tracts up to 24 hours after injection. Hepatocellular disease was characterized by delayed liver clearance and delayed visualization of the biliary and gastrointestinal tracts. There were no toxic or other untoward effects in any patients.     

Photodynamic therapy for premalignant lesions in DMBA-treated hamsters: a preliminary study

PURPOSE: Photodynamic therapy (PDT) involves the selective destruction of neoplastic cells through the activation of a photosensitizer by light. We have previously shown that the photosensitizer Photofrin (porfimer sodium) is selectively accumulated in transformed lesions destined to become malignant, but not yet definable histologically as precancers. The aim of this investigation was to determine if this premalignant tissue could be selectively destroyed by systemically administered Photofrin activated by 630 nm red light via an argon dye laser. MATERIALS AND METHODS: The carcinogenic model used was the DMBA (9, 10 dimethyl 1,2 benzanthracene)-treated hamster cheek pouch. The animals were treated with 0.5% DMBA in acetone thrice weekly for 6 weeks (experiment I, premalignant lesions), or 12 weeks (experiment II, malignant lesions). Ten animals were in experiment I; nine animals were in experiment II. These were divided into experimental and control subgroups. The 6-week experimental group received PDT and CO2 laser incision into the DMBA-treated area. The CO2 laser was used as a promoter of neoplasia in a field that had already undergone initiation from the DMBA treatment. The control groups received either CO2 laser incision alone into the DMBA-treated field or CO2 laser incision and argon pumped dye laser treatment (without Photofrin). The 12-week experimental group received PDT after CO2 laser excision of tumors. The controls received CO2 excision alone, or CO2 excision combined with postoperative hyperthermia. RESULTS: One hundred percent (three of three) of cheeks in experiment I receiving PDT developed necrosis of the treated area within 24 to 48 hours, but 0% (0 of three) subsequently developed tumors. No necrosis was seen in control cheeks receiving Photofrin without irradiation (0 of four) or irradiation without Photofrin (0 of six), and 56% (five of nine) of cheeks exposed to identical carcinogenic stimulus, without PDT, developed tumors (n = 9). In experiment II, 0% (0 of six) of cheeks receiving postoperative PDT developed tumor recurrence. In experiment II controls, 50% (three of six) of cheeks that underwent excision and hyperthermia developed tumor recurrence. In cheeks treated only with CO2 laser excision of tumors, a recurrence rate of 67% (four of six) was noted. These results were found to be statistically significant by the Student t-test on the binomial distribution (P < .01). One animal was treated with DMBA for 6 weeks, administered Photofrin, and the right cheek was irradiated and the animal was left for 30 weeks. The irradiated cheek epithelium necrosed but no cancer developed, whereas the positive control cheek developed a large cancer. CONCLUSION: These results suggest that photodynamic therapy possesses significant potential in elimination of premalignant tissue. This could be beneficial in treating potentially premalignant lesions such as oral leukoplakia, and useful as adjunctive therapy in removal of areas of field cancerization adjacent to surgical sites.     

The human gallbladder increases cholesterol solubility in bile by differential lipid absorption: a study using a new in vitro model of isolated intra-arterially perfused gallbladder

In this study, we first developed and validated a new in vitro isolated, intra-arterially perfused, gallbladder model and then applied the method to investigate the absorption of biliary lipids by the gallbladder wall and the effect of this process on the composition of human bile. Oxygenated and glucose-added buffer was perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with H3-cholesterol and C14-palmitoyl-linoleoyl-phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. Organ viability was demonstrated by stable lactate dehydrogenase (LDH) organ release and oxygen consumption throughout the experiments. In the pig, disappearance rates of lipids from bile were similar in vitro and in vivo, demonstrating the validity of the isolated in vitro model for functional studies. By applying our in vitro isolated preparation to the human gallbladder, we found that 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. As a consequence, at the end of the experiments, cholesterol (P < .05) and phospholipid (P < .05) molar percentages were significantly reduced, while the bile salt (P < .05) molar percentage was significantly increased with respect to values at the beginning of the studies. Our findings are of pathophysiological relevance and support the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile.     

Quantitative estimations of the contribution of different bile acid pathways to total bile acid synthesis in the rat

BACKGROUND & AIMS: Cholesterol degradation to bile acids occurs via "classic" or "alternative" bile acid biosynthetic pathways. The aim of this study was to assess the contributions of these two pathways to total bile acid synthesis in vivo. METHODS: Rats with biliary fistulas were infused with squalestatin for 24 and 48 hours; specific activities of cholesterol 7 alpha-hydroxylase (C7 alpha H) and sterol 27-hydroxylase (S27H) and rates of bile acid synthesis were determined. RESULTS: Continuous squalestatin infusion (15 micrograms/h) decreased C7 alpha H specific activities to 4% and 12% of paired biliary fistula controls at 24 and 48 hours, respectively (P < 0.05) without any changes in S27H specific activities (82% and 95% of controls). At 24 hours, bile acid synthesis decreased to 43% (P < 0.05) but returned to 87% at 48 hours (P = NS). Cholic acid synthesis decreased at 24 hours but returned to control levels at 48 hours. Similar changes in C7 alpha H, S27H, and bile acid synthesis were observed in primary rat hepatocytes after addition of squalestatin (1.0 mumol/L). CONCLUSIONS: In the face of persistent suppression of C7 alpha H and the classic pathway, an alternative pathway becomes a main pathway of bile acid synthesis capable of generating cholic and chenodeoxycholic acids. The observed induction of bile acid synthesis via an alternative pathway or pathways represents an important mechanism for maintenance of cholesterol homeostasis in the rat.     

Effective mechanisms of ATX-S10, a new photosensitizer, on HeLa tumors in nude mice

Photodynamic therapy (PDT) is a treatment modality that utilizes a photosensitizing drug activated by laser generated light. PDT is effective for oncologic applications. Many cancer patients have undergone a hematoporphyrin derivative (HpD)-mediated PDT. The HpD showed a side effect causing prolonged cutaneous photosensitivity. But ATX-S10, a new photosensitizer, provides rapid plasma and tissue clearance, comparable photoactivation efficiency, and superior light absorption of visible red. In this study, the tumor rejection mechanisms of PDT using ATX-S10 on HeLa tumors in nude mice were investigated with morphological and fluorometric methods. The mice were intracutaneously inoculated with HeLa cells, 5 x 10(5) or 1 x 10(7) cells. When tumors grew to about 10-12 mm in diameter, mice were intraperitoneally administered ATX-S10, 30 mg/kg, and 2 hours later the ATX-S10 in tumors was indirectly measured by a fluorometric machine, PMA-10 (Hamamatsu Photonics K. K.) and the tumors were irradiated by Optical Parametric Oscillator (Hamamatsu Photonics K. K.) tuned to a wave length at 670 nm, 5 mJ/pulse, 100 J/tumor. Before and after the irradiation, the effective mechanisms of PDT with ATX-S10 were studied by histological and ultrastructural approaches. The results showed occlusive thrombi in the microvasculature of the tumors and tumor cell death. These occlusive thrombi were observed within one hour after PDT at both light and electron microscopy levels, and were more remarkable as time passed after PDT. Therefore, the morphological studies of PDT with ATX-S10 suggested that the rejection mechanisms occurred mainly as a result of the destructive changes of the microvasculature in the tumors first, and secondly or simultaneously, tumor cells were destroyed through necrosis, and finally the tumors were rejected.     

Gallbladder dyskinesia in acalculous biliary colic

AIMS: To test the hypotheses that: 1) postprandial gallbladder emptying might be abnormal in patients with acalculous biliary colic; and 2) that hymecromone, a drug that increases bile flow and relaxes biliary muscle might improve symptoms. METHODS: Twenty-four women were studied. Twelve patients presented recurrent biliary type of pain in the absence of gallstones, and persistence of contrast in the gallbladder, 24 hours after an oral cholecystogram, suggesting cholesterolosis. Twelve healthy volunteers used as controls had no symptoms and the gallbladder was not visible the day after an oral cholecystogram. Gallbladder emptying induced by an oral meal was studied with ultrasound in both groups before and after the administration of hymecromone. RESULTS: Gallbladder volumes were similar in both groups (23 +/- 10 ml vs. 31 +/- 11 ml; NS). Gallbladder emptying in the control group was simple exponential (R2 = 0.948 +/- 0.059) with a rate of 32 +/- 16 minutes. Gallbladder emptying was bimodal in patients. Gallbladder volume increased to 120% in the first 10 minutes, to empty exponentially thereafter (R2 = 0.964 +/- 0.040. Rate: 29 +/- 22 minutes). This resulted in a marked reduction in gallbladder volume emptied at 40 minutes when compared to controls (30% +/- 27% vs. 54% +/- 14%; p = 0.012). Pretreatment of controls with hymecromone induced an emptying pattern similar to that of patients. In patients, emptying after hymecromone did not change but symptoms were improved. CONCLUSIONS: An abnormal emptying pattern of the gallbladder was identified in patients with acalculous biliary colic, and hymecromone was partly effective to reduce pain, despite a minor effect on gallbladder emptying pattern.     

Formation and inhibition of lysolecithin in human gallbladder bile

Homogenized human gallbladder epithelium was incubated at 37 degrees C with 14C-lecithin in diluted gallbladder bile. During the incubation, lecithin was transformed to lysolecithin. The reaction rate was higher at pH 4.5 than at pH 7.0. No degradation of lecithin occurred if the reaction mixture did not contain the homogenate. Lysolecithin was mixed with red blood cells in (a) diluted human gallbladder bile and (b) 0.15 M saline. The surface activity in the different systems was then assessed from the amount of hemoglobin recovered in the red cell pellet after centrifugation. In human bile, 500 mug lysolecithin/ml did not affect the amount of hemoglobin recovered whereas in saline, concentrations exceeding 25-30 mug/ml affected the red blood cells such that no hemoglobin was pelleted by the centrifugation. Lysolecithin was further studied for effect upon lecithin-3H-cholesterol-dicetylphosphate liposomes containing 14C-glucose. The surface activity of lysolecithin was assessed from the distribution of 3H- and 14C-activity after centrifugation. Although 500 mug lysolecithin/ml increased the non-sedimented 14C-activity, 5000 mug lysolecithin/ml was necessary to decrease significantly the amount of sedimented 3H-activity. The results are interpreted such that phospholipase A activity from the gallbladder epithelium, if released into the gallbladder bile, may generate lysolecithin from lecithin. However, the surface activity and, thus, the inflammatory mediating activity of lysolecithin is inhibited by components in the gallbladder bile, possibly lecithin.     

Comparison of photodynamic therapy with cryotherapy in the treatment of Bowen's disease

The efficacy and suitability of photodynamic therapy (PDT) was compared with that of cryotherapy in the treatment of 40 lesions of Bowen's disease. Lesions were randomized to receive either cryotherapy with liquid nitrogen, or PDT using a portable desktop lamp incorporating a 300 W xenon short arc discharge source. A porphyrin precursor, 5-aminolaevulinic acid (5-ALA), was applied topically 4 h before irradiation in the PDT group. Each lesion received 125 J/cm2 at a fluence rate of 70 mW/cm2. All patients were reviewed at 2-monthly intervals and treatments repeated if required. Cryotherapy produced clearance in 10 of 20 lesions after one treatment, the remaining 10 lesions requiring two or three treatment applications. PDT resulted in clearance of 15 of 20 lesions after one treatment and of the remaining five lesions after a second treatment. The probability that a lesion cleared after one treatment was greater with PDT than cryotherapy (P < 0.01). Cryotherapy was associated with ulceration (five of 20), infection (two of 20) and recurrent disease (two of 20); no such complications occurred following PDT. PDT using a non-laser light source and topical 5-ALA appears to be at least as effective as cryotherapy in the treatment of Bowen's disease with fewer adverse effects.     

Photodynamic laser therapy of basal-cell carcinoma of the lid

Photodynamic therapy (PDT) remains an experimental approach for the treatment of small, mainly superficial malignant tumors. When given intravenously, hematoporphyrin derivative (HpD) selectively photosensitizes tumor tissue. Activated by light of 630 nm wave-length, HpD leads to tumor necrosis. This paper presents the results of PDT to eyelid basal-cell carcinomas in 21 patients. All lesions primarily responded to the treatment and became necrotic. A generalized photosensitization lasting for more than 4 weeks was seen in all patients. In five patients, lid malformations due to scar formation were noted, being marked in three cases. Ten patients showed a recurrence of tumor after 3-12 months. At present, PDT has no advantage over well-established therapies for basal-cell carcinomas of the eyelid.     

Extracellular matrix deposition, lysyl oxidase expression, and myofibroblastic differentiation during the initial stages of cholestatic fibrosis in the rat

Early studies showed that during hepatic fibrosis induced by bile duct ligation, fibroblasts within the portal tracts proliferate and express alpha-smooth muscle (SM) actin, suggesting that they may be involved in the deposition of extracellular matrix components in cholestatic fibrosis. Thus, we investigated the deposition of extracellular matrix components (laminin, fibronectin EIIIA, collagen I and IV, procollagen III, elastin, tenascin) as well as the expression of lysyl oxidase and of alpha-SM actin in the portal zone at 24, 48, and 72 hours and 7 days after ligation of the common bile duct. Rat liver tissues were processed for immunofluorescence, in situ hybridization, immunohistochemistry, and for electron and immunoelectron microscopy. At all times examined after bile duct ligation, laminin was observed essentially in the basal membrane of vessels and portal ductules. In sham-operated animals, the fibronectin EIIIA was present exclusively in vessels; at 24 hours postinjury, fibronectin EIIIA expression appeared in both the portal zone and along sinusoids. Two days after ligation, increased expressions of collagen I and IV, procollagen III, and elastin were observed within the portal zone, compared with sham-operated animals. The deposition of these components increased thereafter. Tenascin expression increased soon after bile duct ligation in stroma surrounding proliferating ductules, reaching a maximum at 48 hours; thereafter, expression was restricted to the periphery of proliferating ductules. By in situ hybridization, procollagen I and tissue inhibitor of metalloproteinase-1 mRNA expression was greatly increased in periductular areas at 24 hours postligation and remained elevated throughout the experiment. At 24 hours, a strong reactivity for lysyl oxidase appeared in the portal zone, and, as in controls, alpha-SM actin expression was restricted to vascular SM cells. In the stroma adjacent to proliferating ductules, alpha-SM actin appeared at 48 hours, and the number of alpha-SM actin-positive cells increased until the 7th day. Lysyl oxidase staining increased until 72 hours after bile duct ligation, when it was located in areas surrounding the myofibroblastic cells. At 7 days, lysyl oxidase expression was restricted around myofibroblastic cells present at the periphery of the reactive tissue and appeared to extend into the surrounding parenchyma. These results show that after bile duct ligation, extracellular matrix deposition, and lysyl oxidase expression occur very early in portal connective tissue surrounding proliferating ductules, and precede myofibroblastic differentiation, ie, alpha-SM actin expression. In addition, the data are compatible with the suggestion that in the bile duct ligation model, myofibroblastic differentiation represents an adaptive response to modification of the extracellular matrix environment.     

Morphological features and healing of stress ulcers induced by alcohol and restraint

Gastric mucosal damage was produced in 130 mice by forced ingestion of alcohol, followed by restraint in the cold. Animals were killed at intervals from six hours to 27 days following the stress. Linear hemorrhagic erosions and diffuse hemorrhagic patches were found grossly, predominantly in the glandular portion of the stomach, in about 75% of the animals 6 to 24 hours after stress. At later time intervals, the linear mucosal depressions persisted; these became progressively less prominent with time but substantial numbers were still readily discernible three weeks after injury. Microscopical examination of the fresh lesions showed a variable amount of mucosal necrosis and acute inflammation. Epithelial regeneration became prominent after three days and persisted for several weeks. The first regenerating epithelial cells were primitive, cuboidal elements followed by mucous cells, parietal cells, and zymogen cells, in that order. The morphological features and evolution of the lesions were similar to that described in human stress ulcers.     

Effects of octreotide on biliary lipid composition and occurrence of cholesterol crystals in patients with acromegaly. A prospective study

Treatment with the somatostatin analog octreotide is associated with increased gallstone formation. The mechanism of formation of these stones is unclear. The purpose of this study was to examine the effect of a three-month treatment with octreotide on biliary lipid composition and the occurrence of cholesterol crystals in patients with acromegaly. Thirteen patients with active acromegaly, aged 24-76 years, received octreotide (100 micrograms three times daily) for three months. Fasting gallbladder bile was obtained during upper gastrointestinal endoscopy after ceruletide stimulation. Bile was studied before and at the end of the treatment period (N = 7), only before (N = 4), or only at the end of treatment (N = 2). Before treatment, all bile samples but one were supersaturated with cholesterol. However, none contained cholesterol crystals on microscopic examination. At the end of the treatment period, all but two samples were supersaturated with cholesterol. Three of nine samples contained cholesterol crystals, a proportion significantly higher than before treatment. The relative proportions of bile acids, cholesterol, and phospholipids, and the mean cholesterol saturation index were not different before and during treatment. Follow-up ultrasonography showed the occurrence of gallstones in four patients, including the three patients who had cholesterol crystals. We conclude that: (1) fasting gallbladder bile of patients with acromegaly is frequently supersaturated with cholesterol; (2) treatment with octreotide does not increase cholesterol saturation index, but may induce the occurrence of cholesterol crystals. The data are consistent with the view that gallstones induced by octreotide are cholesterol stones and suggest that the drug may impair gallbladder motility and/or decrease cholesterol nucleation time.     

Pharmacological characterization of nociceptin receptor: an in vitro study

Pancreatic cancer is difficult to treat, even for tumours localized to the pancreas. Photodynamic therapy (PDT) is a non-thermal technique for producing localized tissue necrosis with light after prior administration of a photosensitizing drug and it could have a role in the local treatment of these cancers. We studied PDT in a transplanted cancer in the hamster pancreas using the photosensitizer mTHPC (meta-tetrahydroxyphenylchlorin). Fluorescence microscopy showed maximum levels of mTHPC in normal pancreas 2-4 days after sensitization and in tumour at 4-5 days. For PDT, animals were given 0.1 or 0.3 mg kg(-1) mTHPC and the tumour was treated at laparotomy 2 or 4 days later with red light (50 J at 650 nm, continuous or fractionated) delivered via a single fibre touching the tumour surface. The maximum zone of tumour necrosis (seen 3 days after PDT) was 8.7 mm in diameter with continuous irradiation, increasing to 12.4 mm with light fractionation (four equal fractions with 3 min between fractions). The main complication was sealed duodenal perforation, seen in 3 of 16 animals, probably due to inadequate shielding of the duodenum from the light. The duodenal problems seen in hamsters are unlikely to cause trouble in the much thicker human duodenum. PDT tumour necrosis in this animal model has now been shown with a range of photosensitizers, but mTHPC is attractive as it is likely to produce the largest volumes of necrosis around each treatment point with short light exposure times. This technique could have a role in the treatment of localized cancers of the pancreas in patients unsuitable for surgery and can now be considered for preliminary clinical trials.     

Photodynamic therapy of the ciliary body with tin ethyl etiopurpurin and tin octaethyl benzochlorin in pigmented rabbits

BACKGROUND AND OBJECTIVES: The authors used a pigmented rabbit model to investigate two photosensitizers, tin ethyl etiopurpurin (SnET2) and tin octaethyl benzochlorin (BNZ 203), to determine their potential for creating ciliary body injuries during photodynamic therapy (PDT). MATERIALS AND METHODS: The biodistribution of SnET2 (n = 10) and BNZ 203 (n = 9) was studied by fluorescence microscopy using a low light detection system, based on charged-coupled device photography, with digital image processing at 1 and 24 hours after injection. PDT with SnET2 (n = 8; 664 +/- 7-nm light; 75 mW/cm2; 50 or 100 J/cm2; 1-mm spot size) and BNZ 203 (n = 6; 689 nm; 75 mW/cm2; 50 or 100 J/cm2; 1-mm spot size) was performed at 24 hours post-injection. The control subjects for SnET2 (n = 5) and BNZ 203 (n = 3) were given a maximal light dose (100 J/cm2). RESULTS: Both photosensitizers demonstrated an intravascular distribution at 1 hour that shifted to a ciliary body distribution at 24 hours (SnET2 much greater than BNZ 203). In addition, the SnET2 demonstrated suborgan localization to the nonpigmented ciliary body epithelium. Both photosensitizing agents were able to produce selective injury to the rabbit ciliary body (SnET2 much greater than BNZ 203), with evidence of a small component of thermal damage (SnET2 greater than BNZ 203). CONCLUSIONS: PDT with SnET2 or BNZ 203 can produce selective injury to the pigmented rabbit ciliary body. The nonpigmented ciliary body epithelium exhibits selective retention of SnET2. This finding warrants further investigation.     

Formation of varanic acid, 3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid in Bombina orientalis

Varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestanoic acid; 24-OH-THCA) is almost the sole component of bile acids in the bile of Bombina orientalis. To examine in the mechanism of the formation of 24-OH-THCA, radiolabeled (25R)- and (25S)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholestanoic acids [(25R)- and (25S)-THCA] and (24E)-3 alpha, 7 alpha, 12 alpha-trihdroxy-5 beta-cholest-24-enoic acid (delta 24-THCA) were administered intraperitoneally to B. orientalis, gallbladder bile was collected after 24 h, and bile acids were subsequently extracted. Then the bile acids were analyzed by means of radio thin-layer chromatography and radio high-performance liquid chromatography after conversion to p-bromophenacyl ester derivatives. Although delta 24-THCA was not converted to 24-OH-THCA, (25R)-THCA and (25S)-THCA were transformed to (24R,25R)-24-OH-THCA and (24R,25S)-24-OH-THCA, respectively. These results strongly suggest that 24-OH-THCA was transformed via direct hydroxylation of the saturated side chain of THCA, not via hydration to an alpha, beta-unsaturated acid, delta 24-THCA, in B. orientalis.     

Effect of photodynamic therapy on the healing of a rat skin flap and its implication for head and neck reconstructive surgery

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) may as adjuvant therapy by used to reduce tumor recurrence in the head and neck with surgery, given intraoperatively after resection. A concern with the use of intraoperative PDT is the possible effect on wound healing, especially on the healing of myocutaneous skin flaps, which are widely used to reconstruct defects following resections for head and neck cancer. STUDY DESIGN/ MATERIALS AND METHODS: A flap, based on the inferior epigastric artery, was prepared in thirty male Lewis rats. Group I did not receive any further treatment but the wound was left open for 20 minutes. Group II was injected with 5mg/kg Photofrin, 48 hours prior to the operation and also did not receive any further treatment. The wound bed and wound borders of group III were treated with 630nm light of different dosages, delivered by an argon dye laser. Animals in group IV received 5mg/kg Photofrin 48 hours prior to the operation and their wound beds were treated with the same light dosages as group III. After the treatment all flaps were replaced into the wound bed and the incisions were closed. Biopsies for histological analysis were taken at several time points; and on day 21, biopsies for wound tensile strength measurements were taken. RESULTS: The wound healing in group I, II, and III appeared normal and there were no differences seen between these groups. Also, the tensile strength did not differ significantly. The flaps of group IV showed serous effusion, epidermal necrosis, and weaker tensile strength (P = .04 and .02 for the light doses of 50 J/sq cm and 75 J/sq cm respectively) at a specific time point. CONCLUSION: The results of this study demonstrate that PDT given immediately before flap reconstruction will result in delayed wound healing. These results should be considered when contemplating the use of PDT as adjuvant intraoperative therapy for tumor surgery requiring flap reconstruction after ablative surgery.