Duodenobiliary Reflux And Intestinal Permeability Increased May Take Part In The Pathogenesis Of Cholelithiasis

IntroductionThe recurrence rate of choledocholithiasis after treatment ranges from 6.4% ~ 18%. It varies depending on the type of the stone. Some factors may take part in the recurrence of choledocholithiasis, such as sphincter of Oddi stricture, stone remnant, chronic cholangitis, biliary bacterial infection, bile duct stricture, unhealthy diet habit, unsuitable treatments or unspecialized performance, etc. As the origin of biliary bacteria, it is generally accepted to from the intes? tine, either via major duodenal papilla, or penetrating intestinal mucosa. However , the evidence of biliary bacteria' s origin is not enough. Therefore, the aim was to find out whether duodenobiliary reflux existed in patients undergone cho-ledocholithotomy plus T-tube drainage, and to assess the sphincter of Oddi motility by choledochoscope manometry.Materials and methodsMaterialsFifty-one patients undergone choledocholithotomy plus T-tube drainage were studied, their mean age was 55 years(27 ~71 years),thirty-six of which were women. Their mean post-operation time was 59 days (45 ~ 92 days). After observing duodenobiliary reflux, they were divided into two groups: reflux group (duodenobiliary reflux positive) and control group (duodenobiliary reflux negative ) , thirty-three of which were selected randomly and double-blind to assess the sphincter of Oddi motility by choledochoscope manometry.Observing duodenobiliary refluxThe patients were asked to fast overnight prior to starting the test. One mil-liliter of water containing 185MBq(5mCi) of technetium-99m diethylenetriamine-pentaacetatic acid (99mTc-DTPA, Mr549000) was injected into the patients' mouth. They then consumed 240 ml of water and lay on back immediately. A 2h bile collection was obtained through the T-tube beginning at the time of ingestion of the tracer. The presence of free pertechnetate was checked by radiochromatography prior to administration to the patients. All the samples employed contained less than 1% of free pertechnetate. To determine the duodenobiliary reflux, a 20 ml of the 2h bile collection was performed technetium counts using RM905 radioactivity meter. Hie principle is that bile could be detected radioactivity , if reflux existed.Sphincter of Oddi manometryA triple lumen polyethylene manometry catheter of 200cm length and 1. 7 mm outer diameter was used for manometry. The side holes in the distal end were located 2mm apart. Each catheter lumen was infused with sterile water at a flow rate of 0. 5ml/min by a minimally compliant hydraulic capillary infusion system. PC polygraph HR (Swedish CTD-Synetics medical company) and relevant program were used to record and analyze the tracings. The manometry was performed after removing all the stones in the Common bile duct. The catheter was introduced via side-pore of choledochofiberoscopy into duodenum directly, when the tracings of the pressure were stable, duodenal pressure-curve was recorded. It was then withdrawn in a stepwise fashion, the position of catheter in the sphincter was confirmed by the characteristic pressure changes seen on the screen. It also could be confirmed by directly observation through choledochofiberoscopy. The SO and common bile duct motility tracings were recorded respectively.ResultsDuodenobiliary refluxSixteen bile samples exhibited radioactivity of all the 51 patients undergone choledocholithotomy plus T-tube drainage, which means the morbidity of duodenobiliary reflux among these people is about 31% (16/51). The mean techneti-um counts of the bile from the 16 reflux positive patients was 209.5 ±264. OKBq (counting performed at the second hour after ingested the isotope). None of the rest 35 bile samples had been detected any radioactivity.Manometry of SOThe sphincter of Oddi' s basal pressure (7. 2 ±3. 9mmHg) , amplitude (53.5 ±24. 5mmHg), common bile duct's pressure(5. 1 ±1.6 mmHg)in the reflux group is significantly lower than in the control group (the value is 14.7 ± 11.0,117.2 ±65.641.5 ±7.4 mmHg respectively,P < 0.05} .while there is no significant difference in the frequency of contractions, duration of contractions or duodenal pressure between the two groups.DiscussionReflux in digestive tract is an abnormal phenomenon, duodenogastric reflux and gastroesophageal reflux can often be found in clinics. Besides, duodenobili-ary reflux and pancreatobiliary reflux are related to cholangitis and common bile duct cyst, respectively. If duodenobiliary reflux happens, duodenal fluid ,intestinal bacteria and endotoxin, or food could get into biliary tract, all of which could lead to cholelithiasis. Therefore duodenobiliary reflux may be a factor of cholelithiasis pathogenesis.Techniques employed to evaluate duodenobiliary reflux include: oral hypaque following X-ray examination, oral soda following sonographic examination and biliary scintigraphy after ingested isotope labelled large molecular weight substance, such as 99mTc-DTPA. Their common characteristics are that all of them need to use complex and expensive medical machine, and some of the results need specialists to understand. The individual determination varies greatly, and their sensitivity is not high. It is hard to evaluate quantitily. We firstly employ 99mTc-DTPA to determine duodenobiliary reflux in patients undergone choledocholithotomy plus T-tube drainage. The only machine used in this kind of method is RM905 radioactivity meter. Since the high molecular weight of 99mTc-DTPA ingested has not any other pathway to get into the bile and is hard to penetrate the intestinal mucosa . If we have detected radioactivity in the pa-tients' bile, we can infer they are duodenobiliary reflux positively. The method is simple, low-cost and safe, and it is easy to repeat the research within short period for the short half-life of the technetium. The results can be easily understood by common people (even they may be not familiar with iconography diagnosis). It has a high specificity (we could say 100% ), and the sensibility is still unknown since the occurrence of duodenobiliary reflux may be influenced by some factors, such as the state of the sphincter of Oddi. This means the real duodenobiliary reflux among patients undergone choledocholithotomy may be more larger than our results.This research firstly employed 99mTc-DTPA to detect duodenobiliary reflux in patients with T-tube drainage, and found that the morbidity of duodenobiliary reflux among these people is 31% ,and of all the ingested dose of nucleus, the refluxed dose is about 1.1%. Though there are no healthy subjects as control :group in this study, duodenobiliary reflux should not be existed in healthy people. Since normal spincter of Oddi posseses the effect of non-return valve, and can maintain biliary pressure, regulate bile excretion, prevent duodenobiliary reflux occuring. Whether the high morbidity of duodenobiliary reflux among patients with T-tube drainage is related to sphincter of Oddi dysfunction? Manometry is the gold standard to study the sphincter of Oddi' s function presently, and has important significance in assessing the biliary kinetics and sphincter of Oddi' s function. So we selected 33 patients randomly and double-blind from all the subjects to perform sphincter of Oddi' s manometry, and found that sphincter of Oddi' s contraction amplitude, basal pressure and common bile duct pressure in the reflux group were significantly lower than that in the control group. While there were no significant difference in the duodenal pressure, sphincter of Oddi' s duration and frequency between the two groups. After deeply analysed, we found that the pressure difference between common bile duct and duodenum (CBDP-DP) in the reflux group is significantly lower than in the control group.Normal sphincter of Oddi can maintain biliary pressure, and make common bile duct' s pressure higher than duodenal pressure, and duodenobiliary reflux can't occur. Morover, Calabuig et al. found that even the perfusion pressure to the duodenum of opossum was as high as 100cmH2O, duodenobiliary refluxcould not be observed. In this research, we found that the pressure difference between common bile duct and duodenum in the reflux group was decreased. It is the manifestation of sphincter of Oddi dysfunction, the result is duodenobiliary reflux occurred.The current investigation firstly reports that duodenobiliary reflux existed in patients undergone choledocholithotomy. This factor may play a role in the high recurrence rate of choledocholithiasis. Because intestinal bacteria and endotoxin are more easily to reach the hepatobiliary system in patients with duodenobiliary reflux. Some bacteria in hepatobiliary system can produce beta-glucuronidase and phospholipase A, as we known, which can respectively hydrolyze conjugated bilirubin and lecithin . The products, unconjugated bilirubin and palmitic acid , precipitate with calcium to form gallstone. Endotoxin could activate the hepatocytes, biliary epithelial cells or leukocytes in the bile to release endogenous beta-glucuronidase by cytotoxin effect or other mechanism. Ho et al. found endogenous beta-glucuronidase also could play an important role in the pathogenesis of pigment stones.Aside from the bacteria and endotoxin, duodenal fluid and food may also get into bile duct through reflux. In the study in vitro, the addition of duodenal fluid into lithogenic bile can promote gallstone formation. In addition, the ingested biliary foreign bodies such as fish bone and cherry stalk etc. may be important in the nucleation. Mucin, calcium bilirubinate, bacteria and other substance in the bile may then aggregate around the nucleus, forming gallstone. All these clarify that duodenobiliary reflux plays a role in the pathogenesis of cholelithiasis.In summary, duodenobiliary reflux have been observed to occur in patients undergone choledocholithotomy plus T-tube drainage. The mechanism is still not known clearly, may be the result of the pressure difference decreased between common bile duct and duodenum, i. e. sphincter of Oddi dysfunction, while they may play an important role in the high recurrence rate of choledocholithiasis. The method employed in this study is easier to procedure, lower costs and greater availability. Long-term studies employing this method to investigate larger samples should be carried out, and the research about how duodenobiliaryreflux developed remains to be clarified.ConclusionOral 99mTc-DTPA is an effective method to determine duodenobiliary reflux; Duodenobiliary reflux existed in patients with T-tube drainage, the morbidity a-mong these people is about 31% ; Duodenobiliary reflux is relation to the significant decrease of sphincter of Oddi' s contraction amplitude, basal pressure, and the pressure difference between common bile duct and duodenum, while is not relation to sphincter of Oddi ' s contraction frequency, duoration or duodenal pressure; Duodenobiliary reflux may take part in the pathogenesis of choledocho-lithiasis.IntroductionAs the origin of biliary bacteria, it is generally accepted to come from the intestine, either via major duodenal papilla, or penetrating intestinal mucosa, while the evidence of biliary bacteria's origin is not much. In normal condition, intestinal mucosa posseses barrier function to bacteria, endotoxin, food antigen, etc. So large molecular substance, such as bacteria, is hard to penetrate the intestinal mucosa. Articles about the intestinal permeability in patients of cholelithiasis were seldom, therefore this research is to find out the state of intestinal permeability in these people.Materials and methodsMaterialsWe studied 56 patients of cholelithiasis. They were divided into cholesterol stone group ( CS group, n = 15 ) and pigment stone group ( PS group, n = 41) based on the cross section of the stone during operation. For the assessment of normal intestinal permeability values, seventeen healthy subjects acted as controls (C group,n = 17). All were free of gastrointestinal disorders and known renal disease.MethodsAll the subjects employed, including the patients and controls, administered 99mTc-DTPA 185MBq(5mCi) orally after fasting overnight and emptying their bladder. A 24h urine collection was obtained after the ingestion of the tracer. To calculate the urinary excretion of 99mTc-DTPA, a 10 ml aliquot of the 24h urine collection was counted along with a 10 ml aliquot of a 1; 1000 dilution of the standard dose of 99mTc-DTPA. The calculation formula is as follows: urinaryexcretion of 99mTc-DIPA = [ (urinary counting-background) × urinary volumn/ (standard counting-background) ×1000] ×100%.ResultsThe mean percentage of the total ingested dose of 99mTc-DTPA excreted in a 24h urinary excretion was 5.0 ±3.6% in CS group, 10.5 ±6.9% in PS group, and 4.5 ± 3.4% in controls. The intestinal permeability in PS group demonstrated significantly increased values compared to controls(P <0.05). While there is no significant difference between CS group and C group (P >0.05).DiscussionIn normal condition, intestine mucosa posseses barrier function, besides the basic function of digestion and absorption. This can refrain intestinal bacteri-a, endotoxin or other harmful substance from entering blood. So intestinal mucosa doesn ' t absorb some substance. When the barrier function of intestinal mucosa damaged, the dose of these substance penetrating intestine would increase. Then the intestinal permeability increased. So the high intestinal permeability, or hyperpermeability, is an indirect marker of damaged intestine, which is the theoretical base of this research. The purpose is to compare the intestinal permeability of patients of different kinds of cholelithiasis with healthy subjects. Such article is seldom at present.In this research, intestinal permeability is expressed as the percentage of the ingested dose of 99mTc-DTPA excreted in a 24h urinary collection. The urinary excretion of nonabsorbable ingested carbohydrates such as lactulose or manni-tol, or orally administered polyethylene glycol, or radioactivity isotope labelled large molecular weight substance such as 51Cr-ethylenediamine tetraacetate (51Cr-EDTA) or 99mTc-DTPA, has been employed to assess intestinal permeability. Hyperpermeability or increased intestinal permeability has been investigated to present in patients with gastrointestinal disorders, hepatocirrhosis, and fasting for postoperation. While the intestinal permeability in patients with cholelithiasis oreasily recurrent choledocholithiasis is still unknown. Based on our observation, the hyperpermeability state is present in patients of pigment stone. Higher mean levels of urinary recovery of 99mTc-DTPA were seen in these patients compared with the controls, and this difference is considered significant (P < 0.05).Based on this study, we suspect that certain factor caused the intestinal permeability increased, i. e. the hypofunction of intestinal barrier. Which resulted in intestinal bacteria translocation. Because intestinal bacteria and endotoxin are more easily to reach the hepatobiliary system in patients with hyperpermeability. Some bacteria in hepatobiliary system can produce beta-glucuronidase and phospholipase A, as we known, which can respectively hydrolyze conjugated bilirubin and lecithin . The products, unconjugated bilirubin and palmitic acid , precipitate with calcium to form gallstone. Endotoxin could activate the hepatocytes, biliary epithelial cells or leukocyte in the bile to release endogenous beta-glucuronidase by cytotoxin effect or other mechanism. Ho et al. found endogenous beta-glucuronidase also could play an important role in the pathogenesis of pigment stones.The area of intestinal mucosa is as high as hundreds of square metres, and is the surface system of normal microorganism. This system is made up of four parts of barrier: machine, organism, chemistry and immunology. When one of these barrier is disorganized, pathogenic bacteria and toxin could penetrate the intestinal wall, and invade mesenteric lymphoid tissue, hepatobiliary system or blood, etc.In clinics, we found the poor and malnourished are more easily to trouble choledocholithiasis or intrahepatic lithiasis. One reason may be they are lack in glucaro-1,4-lactone, a β-glucuronidase inhibitor, which potentializes the decon-jugation reaction of bilirubin. Another may be these people intaked low protein and fat, which might have not advantage in the growth and development of the intestinal mucosa. As a result, the intestinal mucosal integrity was damaged and hyperpermeability occurred. Then intestinal bacteria translocated into hepatobiliary system , playing a role in gallstone formation.In summary, employing the method of oral 99mTc-DTPA, we firstly found that the intestinal permeability in patients of pigment stone was significantlyhigher than that in healthy subjects. Hyperpermeability may be a factor of the cause of pigment stone.ConclusionOral 99mTc-DTPA is a convenient, easy-procedure and low-price method to determine intestinal permeability. Intestinal permeability in patients of pigment stone is higher than healthy subjects. While there is no difference between patients of cholesterol stone and healthy subjects. Hyperpermeability may be a factor of the pathogenesis of pigment stone.IntroductionThrough the above-mentioned research, we found duodenobiliary reflux and hyperpermeability of intestine were existed in patients of cholelithiasis. The results are intestinal bacteria and endotoxin translocation. The endotoxin get into hepatobiliary system could activate human leukocytes, leading to release many kinds of cytokine, such as tumor necrosis factor ( TNF) , interleukin (IL-8, IL-10), etc. Whether endotoxin could also activate hepatocytes, epithelial cells of gallbladder and bile duct or leukocytes in bile to release endogenous β-glucuronidase is still not known. The purpose of this research is to investigate the effect of lipopolysaccharide(LPS) on the expression of hepatocyte GUSB mRNA and the activity of serumal β-glucuronidase(β-GD) in mice, and discuss a kind of mechanism of cholelithiasis pathogenesis.Materials and methodsMaterialsAnimals: The strain of mice selected was more sensitive to endotoxin, that was BALB/c mice(6 ~8weeks). Female or male was not restrained, and their body weight were about 20 ~ 25 grams.Reagents: Phenolphthalein-mono-β-glucuronic acid and endotoxin ( Sigma company); Total RNA Extraction System ( Ⅱ ) ; dNTPs; AMV Reverse transcriptase; Oligo-dT15; Rnase-inhibitor; Taq TM (Huamei Organism engineering company ) ; primers of GUSB and β-actin( Boya company ).Instruments: PTC-100 TM Programmable Thermal Controller (MJ Reserch. Inc. u. s. a. } ; universial eletrophoresis apparatus ( Biometra, u. s. a. ); cryo-genie refrigerator (Formas Scien. Inc. u. s. a. ); System of image analysis of infrared-white light transmission meter 10Kodak (Kodak, u. s. a. ). Methods1. Establishing experimental animal model and dividing into groups: Twenty-three mice were randomly allocated to control group (injection of Saline without endotoxin) and LPS group(received dosage of endotoxin was 5 mg/Kg). In the LPS group, after the injection of endotoxin, the mice were put to death by breaking cervical vertebra at the 1/2,2,4,24 hour. Taking blood to anticoagu-late and centrifugate, and the serum was frozen; taking liver into Eppendorf tube which had been treated with DEPC water, and stored at -70℃.2. Enzyme kinetics method to determine the activity of serumal β-glucu-ronidase: 0. 1ml of 0. 01mol/L phenolphthalein-mono-β-glucuronic acid was added into 100μl of serum, then added acetic acid buffer 100μl and regulated the value of pH to 4. 5. The reaction was processed for 24 hours at 38℃. After current cooling, 1. 0ml of glycine-sodium dodecylsulphate (pH = 11.7) was added , 1.5 ml of deionized water was also added and mixed equally. Optical density (O. D. ) was detected at 540nm wave-length. Taking phenolphthalein as standard character, 0 ~ 100μg/ml of concentration series to draw phenolphthalein stand curve.3. Semi-quantity reverse transcriptional — polymerase chain reaction ( RT-PCR) to detect the expression of GUSB mRNA in hepatocytes; Extracting RNA: the total RNA was extracted by Total RNA Extraction System ( Ⅱ) following the instruction. The cDNA was stored at - 70℃. The cDNAs were amplificated with primers of GUSB and β-actin by PTC-100TM Programmable Thermal Controller. The product underwent 2% of agarose gel electrophoresis (60v, 1. 5h), taken β-actin as inter-contrast. The quantity of GUSB mRNA were expressed as a ratio relative to β-actin's by O. D. of their light zone on the gel.Results1. The exist status of mice: The longest time of endotoxin stimulating mice was 24 hours and there were no dealth or abdominal cavity infection occurring.2. The effect of endotoxin on the activity of serumal β-glucuronidase: Non-stimulated serum (the control group) could be determined certain activity of β-GD(2. 14 ± 1. 46 Fishman Unit), in LPS 1/2,2 and 4h group, the activity (3.96 ±0. 88,4. 40 ±0. 55,5. 65 ±0. 58 Fishman Unit,respectively) was all significantly higher than the control's (P < 0.05). While there was no significant difference between the LPS 24h group (3. 85 ± 1.11 Fishman Unit) and the control group.3. The effect of endotoxin on the expression of GUSB mRNA in hepatocytes of mice: In contrast to the control group(0.57 ±0.36) ,the expression of GUSB mRNA in the LPS group is significantly higher after stimulated for 2 and 4 hours (1.10 ±0. 23 and 1. 81 ±0.04, respectively). While there was no significant difference between the LPS 1/2h, LPS 24h group (0. 85 ± 0.29,0.94 ± 0.51, respectively) and the control group.DiscussionThe principle of method employed in this research to determine β-glucu-ronidase is; Tissue β-glucuronidase can hydrolyze phenolphthalein-mono-β-glu-curonic acid at its optimal value of pH(4.5). The products are phenolphthalein and sodium β-glucuronide, and phenolphthalein take on red in alkaline environment. The activity of β-glucuronidase is direct proportion to the colour. So the microgram of phenolphthalein released per hour in 100ml of serum was taken as the activity unit of β-glucuronidase (Fishman Unit) , that is 1 Fishman Unit = 1 μg phenolphthalein /h · 100ml serum.Human β-glucuronidase (EC 3.2.1.31) is a kind of acidic hydrolase and its system name is β-D-Glucuronide glucuronosohydrolase, which can specificly catalyze β-glucuronide bond at the C1 location of glucuronic acid and release glu-curonosic acid, β-glucuronidase is widely distributed in every kind of tissue (such as liver, kidney, spleen, suprarenal gland, thyroid, gastrointestinal mucosa , uterus, preputial gland) , cell (such as erythrocyte, leukocyte, platelet, macrophagocyte, fibroblast) , and body fluid (such as blood, urine, cerebrospinal fluid, bile, vaginal fluid, gastric juice, duodenal fluid). Its intracellular lo-cation is major in lysosome, mitochondrion, microsome. It is a kind of glycoprotein of tetramer, molecular weight is 275000. The activity of β-glucuronidase has great difference between individual. Its real physiologic function in organism is still not known clearly. It may have relationship with metabolism of bilirubin , biotransformation of steroid hormone, hydrolysis of glucuronic acid. It also may take part in the liquefaction of cellular organ and renovation of cell; β-glucu-ronidase released from polymorphonuclear leukocyte possesses important effect in inflammatory tissue damnification and destroying bacteria. Another effect is degradation of glycosaminoglycan by removing β-glucuronylacyl residue at the end of chondroitin sulfate and heparan sulfate in lysosome. The deficiency of β-glu-curonidase can lead to storage of nondegradated glycosaminoglycan, which is mucopolysaccharidosis storage disease type seven (MPS Vtt).Calcium bilirubinate accounts for about 60 percent of brown pigment stone, and 40 percent of black pigment stone. Some cholesterol stone has a core of calcium bilirubinate. That illuminates the pathogenesis of cholelithiasis is tightly correlation to the metabolism of bilirubin. It is well known that Maki considered bacterial β-glucuronidase hydrolyzed conjugated bilirubin, which took part in the formation of pigment stone. That bacteria were not existed in all bile or stone of patients of cholelithiasis suggests nonbacterial factors should also play a role in the cholelithiasis. Since endogenous β-glucuronidase can hydrolyze bilirubin diglucuronide in like manner, it may be relevant to pigment stone. Ostrow et al. considered β-glucuronidase in biliary tract and liver hydrolyzed conjugated bilirubin ? which led to pigment precipition and stone fonnation. Ho found the activity of endogenous β-glucuronidase in bile of brown pigment stone group was significantly higher than that in control group. Furthermore, its activity was strongly positive correlation to the decompound degree of bilirubin. He inferred endogenous β-glucuronidase might play an important role in the formation of pigment stone. Yang B et al. undertook quantity research of endogenous β-glucuronidase in lysosome of hepatocyte in patients of bilirubin calculus by immunoelectron microscope colloid probe technique, and found that endogenous β-glucuronidase was correlation to the formation of bilirubin calculus.Endogenous β-glucuronidase has many kinds of styles, the optimal pH is3. 8, 4.5 and 5.2, respectively, which is much different from the pH value of normal bile. Besides, β-glucuronidase is inhibited by glucaro-1,4-lactone and salt of bile acid. So the activity of endogenous β-glucuronidase in bile is very weak. When infection of biliary tract, hepatocyte, epithelium of gallbladder and bile duct, leukocyte in bile would synthesis more enzyme. The permeability of cell membrane would increase. All these led to the enzyme released to outside of cell increased. Osnes found the level of LPS in bile of common bile duct was closely relation to pigment stone, and inferred that LPS might activate mucous cell of biliary tract and leukocyte in bile to release endogenous β-glucuronidase by cytotoxin effect. If the β-glucuronidase in bile increased to beyond the effect of inhibitor, conjugated bilirubin would be hydrolyzed and pigment stone formed.In this research, there was certain activity of β-glucuronidase in the serum of non-stimulated B ALB/c mice, there was also certain expression of GUSB mR-NA in the hepatocyte. After injection of LPS, both the activity of β-glucuroni-dase in serum and the expression of GUSB mRNA in hepatocyte increased. As for the activity of β-glucuronidase, this increase was observed to start at the 30 minutes after injection of endotoxin (LPS 1/2h group) , and increased to high peak at the 4th hour, then decreased. In 24h group, the enzymatic activity had been no significant difference with the control group. As for the expression of GUSB mRNA, it was also time dependent increase during the 24 hours of LPS stimulating. That is the expression of GUSB mRNA increased firstly, then decreased. However, this increase wasn't significant different with the control group until LPS stimulating for 2 hours. The expression continued to increase at 4h group, and then decreased to the basal level at 24 hour.The mechanism of LPS increasing the activity of β-glucuronidase and the expression of GUSB mRNA may be as follows: LPS combines with CD14 in the surface of hepatocyte and peripheral blood monocyte, which possesses signal transmission function. Through toll like receptor 4 ( TLR4) , transmembrane transduction realizes, making nuclear factor-KB ( NF-kB ) transfer into nuclei from cellular plasm. Thus switches on the gene controlling β-glucuronidase (GUSB gene)to transcript more mRNA. The result is synthesis of β-glucuroni-...