1、Designation: E 1398 91 (Reapproved 2003)Standard Practice forIn Vivo Rat Hepatocyte DNA Repair Assay1This standard is issued under the fixed designation E 1398; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revis
2、ion. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers a typical procedure and guidelinesfor conducting the rat in vivo hepatocyte DNA repair assay. Theprocedure
3、s presented here are based on similar protocols thathave been shown to be reliable (1, 2, 3, 4, 5).21.2 Mention of trade names or commercial products aremeant only as examples and not as endorsements. Othersuppliers or manufacturers of equivalent products are accept-able.1.3 This standard does not p
4、urport to address all of thesafety concerns associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2. Significance and Use2.1 Measurement of chemically ind
5、uced DNA repair is ameans of assessing the ability of a chemical to reach and alterthe DNA. DNA repair is an enzymatic process that involvesrecognition and excision of DNA-chemical adducts, followedby DNA strand polymerization and ligation to restore theoriginal primary structure of the DNA (6). Thi
6、s process can bequantitated by measuring the amount of labeled thymidineincorporated into the nuclear DNA of cells that are not inS-phase and is often called unscheduled DNA synthesis (UDS)(7). Numerous assays have been developed for the measure-ment of chemically induced DNA repair in various cell
7、linesand primary cell cultures from both rodent and human origin(4). The primary culture rat hepatocyte DNA repair assay hasproven to be particularly valuable in assessing the genotoxicactivity of chemicals (8). Genotoxic activity often results frommetabolites of a chemical. The in vitro rat hepatoc
8、yte assayprovides a system in which a metabolically competent cell isalso the target cell. Most other in vitro short-term tests forgenotoxicity employ a rat liver homogenate (S-9) for metabolicactivation, which differs markedly in many important waysfrom the patterns of activation and detoxification
9、 that actuallyoccur in hepatocytes. An extensive literature is available on theuse of in vitro DNA repair assays (9-19).2.2 A further advance was the development of an in vivo rathepatocyte DNA repair assay in which the test chemical isadministered to the animal and the resulting DNA repair isassess
10、ed in hepatocytes isolated from the treated animal (20).Numerous systems now exist to measure chemically inducedDNA repair in specific tissues in the whole animal (4). Theaverage of in vivo assays is that they reflect the complexpatterns of uptake, distribution, metabolism, detoxification,and excret
11、ion that occur in the whole animal. Further, factorssuch as chronic exposure, sex differences, and different routesof exposure can be studied with these systems. This isillustrated by the potent hepatocarcinogen 2,6-dinitrotoluene(DNT). Metabolic activation of 2,6-DNT involves uptake,metabolism by t
12、he liver, excretion into the bile, reduction ofthe nitro group by gut flora, readsorption, and further metabo-lism by the liver once again to finally produce the ultimategenotoxicant (21). Thus, 2,6-DNT is negative in the in vitrohepatocyte DNA repair assay (22) but is a very potent inducerof DNA re
13、pair in the in vivo DNA repair assay (23, 24).Aproblem with tissue-specific assays is that they may fail todetect activity of compounds that produce tumors in othertarget tissues. For example, no activity is seen in the in vivoDNA repair assay with the potent mutagen benzo(a)pyrene(BP), probably bec
14、ause limited tissue distribution and greaterdetoxification in the liver yields too few DNA adducts toproduce a measurable response (3). In contrast, BP is readilydetected in the less tissue-specific in vitro hepatocyte DNArepair assay (11). An extensive literature exists on the use ofthe in vivo hep
15、atocyte DNA repair assay (1-3, 5, 9, 25-33).3. Procedure3.1 Treatment:3.1.1 All personnel must be knowledgeable in the proce-dures for safe handling and proper disposal of carcinogens,potential carcinogens, and radiochemicals. Disposable glovesand lab coats must be worn.3.1.2 Any strain or sex of ra
16、t may be used. The largestdatabase is for male Fischer-344 rats. Young adult animals arepreferred. It is possible that factors such as sex, age, and strainof the rat could affect the outcome of the DNA repair1This practice is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materi
17、als and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Sept. 10, 2003. Published September 2003. Originallypublished in 1991. Last previous edition published in 1998 as E 1398 91 (1998).2The boldface numbers in parentheses ref
18、er to the list of references found at theend of this practice.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.experiments. Therefore, for any one series of experiments(including controls) these variables should be kept constant.3.1.3
19、 Administration is usually by gavage with chemicalsdissolved or suspended in an appropriate vehicle such as wateror corn oil, depending on solubility. An advantage of the assayis that various routes of administration may be chosen. Thus,chemicals may also be administered by intraperitoneal injec-tio
20、n or inhalation or in the diet. For gavage administration, 0.2to 1.0 mL of test chemical solution is administered per 100 gbody weight. Controls receive the appropriate vehicle solution.Stock corn oil should be replaced with fresh monthly.3.1.4 For DNA repair studies, animals may be taken off feedfo
21、r a few hours prior to sacrifice to make the process ofperfusion a little easier with less food in the stomach. Theperiod without food should never exceed 12 h because of thepossibility of altered metabolism or uptake. Water should becontinuously available.3.1.5 Dose selection will depend on the cha
22、racteristics ofeach chemical and the purpose of the experiment. If one isinvestigating whether a chemical can produce a genotoxiceffect in the animal, even at massive doses and by routes ofadministration that may overwhelm natural defense mecha-nisms, then high doses (such as the LD50, or higher) th
23、at donot kill the animal before the 16-h sacrifice point may beemployed. Even in such a case, doses above 1000-mg/kg bodyweight are not recommended. In some instances hepatotoxicityat high doses may result in inhibition of cell attachment orDNA repair. More commonly, the purpose of employing thewhol
24、e animal is to evaluate the genotoxic effects of realisticexposures and routes of administration in the target tissue. Inthis case, doses above 500 mg/kg and the intraperitoneal routeof administration are not recommended. The usual range ofdoses is from 10 to 500 mg compound per kilogram bodyweight.
25、 Target doses with a new compound are usually theLD50 and 0.2 3 the LD50, with 500 mg/kg as an upper limit.The potent mutagen dimethylnitrosamine (DMN) (often usedas a positive control) can be detected with doses as low as 1mg/kg. Normally, an initial range finding experiment is con-ducted using sin
26、gle animals to cover a range of times anddoses. If a positive response is seen, additional experiments areconducted to establish the dose-response relationship. If noresponse is seen, the highest dose(s) is repeated. The finalreport should contain results from at least three animals perdatapoint.3.1
27、.6 Thus far, no examples have been seen of a compoundthat produces a UDS response in female rats but not males. Forthose cases where a comparison has been made, males respondmore strongly than females in this assay. Thus, for the purposeof routine screening only male rats need to be used.3.1.7 Treat
28、ed animals should be maintained in a ventilatedarea or other suitable location to prevent possible humanexposure to expired chemicals. Contaminated cages, bedding,excreta, and carcasses should be disposed of safely accordingto standard published procedures.3.2 Liver Perfusion:3.2.1 Any proven techni
29、que which allows the successfulisolation and culture of rat hepatocytes can be used. Anexample of one such procedure is given in 3.2.2-3.2.17.3.2.2 DMN exhibits a maximum UDS response 1 h aftertreatment. However, the response remains elevated and mea-surable for at least 16 h after treatment. More c
30、ommonly,however, chemicals (for example, 2,6-DNT and2-acetylaminofluorene (2-AAF) show a peak response 12 to16 h post-treatment.The time from treatment to perfusion maybe varied to obtain a time course of induced repair. The routineprotocol for primary screening involves a time point between12 and 1
31、6 h with an optional time point between 1 and 4 h.3.2.3 Anesthetize the rat by intraperitoneal injection with a50-mg/mL solution of sodium pentobarbital (0.2 mL per 100 gbody weight) 10 min prior to the perfusion procedure. Otherproven anesthetics are also acceptable. Make sure that theanimal is com
32、pletely anesthetized, so that it feels no pain.3.2.4 Secure the animal with the ventral surface up onabsorbent paper attached to a cork board. Fold the paper in oneach edge to contain perfusate overflow. Thoroughly wet theabdomen with 70 % ethanol and wipe with gauze for cleanli-ness and to discoura
33、ge loose fur from getting on the liver whenthe animal is opened.3.2.5 Make a V-shaped incision through both skin andmuscle from the center of the lower abdomen to the lateralaspects of the rib cage. Do not puncture the diaphragm or cutthe liver. Fold back the skin and attached muscle over the chestt
34、o reveal the abdominal cavity.3.2.6 Place a tube approximately 1 cm in diameter under theback to make the portal vein more accessible.3.2.7 Move the intestines gently out to the right to reveal theportal vein. Adjust the tube under the animal so that the portalvein is horizontal.3.2.8 Put a suture i
35、n place (but not tightened) in the centerof the portal vein and another around the vena cava just abovethe right renal branch.3.2.9 Perform perfusions with a peristaltic pump, the tubingof which is sterilized by circulation of 70 % ethanol followedby sterile water. Place a valve in the line so that
36、the operatormay switch from the EGTA solution to the collagenase solutionwithout disrupting the flow. Solutions should be kept at atemperature that results in a 37C temperature at the hepaticportal vein.3.2.9.1 A peristaltic pump with a chargeable pump head andsilicone tubing is suitable for perform
37、ing the perfusion.3.2.9.2 Begin the flow of the 37C EGTA solution at 8mL/min and run to waste.3.2.10 Cannulate the portal vein with a 20 GA 114-in.catheter about 3 mm below the suture. Remove the innerneedle and insert the plastic catheter further to about13 thelength of vein and tie in place by the
38、 suture. Blood shouldemerge from the catheter. Insert the tube with the flowingEGTA in the catheter (avoid bubbles) and tape in place.3.2.11 Immediately cut the vena cava below the right renalbranch and allow the liver to drain of blood for 1.5 min. Theliver should rapidly clear of blood and turn a
39、tan color. If alllobes do not clear uniformly, the catheter has probably beeninserted too far into the portal vein.3.2.12 Tighten the suture around the vena cava and increasethe flow to 20 mL/min for 2 min. The liver should swell at thispoint. In some cases gentle massaging of the liver or adjusting
40、E 1398 91 (2003)2the orientation of the catheter may be necessary for completeclearing. At this point the vena cava above the suture may beclipped to release some of the pressure in the liver.3.2.13 Switch the flow to the 37C collagenase solution for12 min. During this period, cover the liver with s
41、terile gauzewetted with sterile saline or WEI (see Annex A1) and place a40-W lamp 2 in. above the liver for warming. It is valuable toscreen each new batch of collagenase to be ensured that it willfunction properly.3.2.14 Allow the perfusate to flow onto the paper and collectby suction into a vessel
42、 connected by means of a trap to thevacuum line. The perfusate should be disposed of as hazardouswaste.3.2.15 After the perfusion is completed, remove the catheterand gauze. Remove the liver carefully by cutting away themembranes connecting it to the stomach and lower esophagus,cutting away the diap
43、hragm, and cutting any remaining attach-ments to veins or tissues in the abdomen.3.2.16 Hold the liver by the small piece of attached dia-phragm and rinse with sterile saline or WEI (see Annex A1).3.2.17 Place the liver in a sterile petri dish and take to asterile hood to prepare the cells.3.3 Prepa
44、ration of Hepatocyte Cultures:3.3.1 Place the perfused liver in a 60-mm petri dish andrinse with 37C WEI (see Annex A1). Remove extraneoustissues (fat, muscle, and so forth).3.3.2 Place the liver in a clean petri dish and add 30 mL offresh collagenase solution at 37C.3.3.3 Carefully make several inc
45、isions in the capsule of eachlobe of the liver. Large rips in the capsule lead to largeunusable clumps of hepatocytes.3.3.4 Gently comb out the cells constantly swirling the liverwhile combing. A sterile, metal, dog-grooming comb with teethspaced from 1 to 3 mm or a hog bristle brush works well.3.3.
46、5 When only fibrous and connective tissue remain,remove and discard the remaining liver. Add 20 mL cold WEI(see Annex A1) and transfer the cell suspension to a sterile50-mL centrifuge tube using a wide-bore sterile pipe. Somelaboratories report successful hepatocyte preparations when3.3.1 through 3.
47、3.8 are conducted with media at room tempera-ture or heated to 37C.3.3.6 Allow the cells to settle on ice for 5 to 10 min until adistinct interface is seen. Carefully remove and discard thesupernatant by suction.3.3.7 Bring the cells to 50 mL with cold WEI (see AnnexA1). Resuspend the cells by pipet
48、ing with a wide-bore pipet.Gently pipet the suspension through a 4-ply layer of sterilegauze into a sterile 50-mL centrifuge tube.3.3.8 Centrifuge the cells at 50 times gravity for 5 min anddiscard the supernatant. Gently resuspend the pellet in ice-coldWEI (see Annex A1) with a wide-bore pipet.3.3.
49、8.1 Some laboratories prefer to keep the cells on iceuntil ready for use, while others keep them at room tempera-ture. Cells should be used as soon as possible, preferablywithin 1 h.3.3.9 Determine viability and cell concentration by themethod of trypan blue exclusion. The preparation should beprimarily a single-cell suspension with a viability of over 60 %for control cultures. With practice and the proper technique,viabilities of about 90 % can routinely be obtained. Attachmentof the cells to the substrate is an active process. Thus, if asufficient number of cells attach to c
