1、Designation: E 1397 91 (Reapproved 2003)Standard Practice forIn Vitro Rat Hepatocyte DNA Repair Assay1This standard is issued under the fixed designation E 1397; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revi
2、sion. 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 vitro hepatocyte DNA repair assay.The procedu
3、res presented here are based on similar protocolsthat have been shown to be reliable (1-6)2.1.2 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 purport t
4、o 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 induced DNA
5、 repair is ameans of assessing the ability of a chemical to reach and alterthe DNA. DNA repair is an enzymatic process that involves therecognition and excision of DNA-chemical adduct followed byDNA strand polymerization and ligation to restore the originalprimary structure of the DNA (7). This proc
6、ess 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)(8). Numerous assays have been developed for the measure-ment of chemically induced DNA repair in various cell linesa
7、nd primary cell cultures from both rodent and human origin(9). The primary rat hepatocyte DNA repair assay developed byWilliams (10) has proven to be particularly valuable inassessing the genotoxic activity and potential carcinogenicityof chemicals (11), (12). Genotoxic activity is often produced by
8、reactive metabolites of a chemical. The in vitro rat hepatocyteassay provides a system in which a metabolically competentcell is itself the target cell for measured genotoxicity. Mostother short-term tests for genotoxicity employ a rat liverhomogenate (S-9) for metabolic activation, which differsmar
9、kedly in many important ways from the patterns ofactivation and detoxification that actually occur in hepatocytes.An extensive literature is available on the use of in vitrohepatocyte DNA repair assays (2, 3, 6, 13-28).3. Procedure3.1 Liver Perfusion:3.1.1 All personnel must be knowledgeable in the
10、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 proven technique which allows the successfulisolation and culture of rat hepatocytes can be used. Anexample of one such procedure is given
11、 in 3.1.3-3.1.20.3.1.3 Any strain or sex of rat 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 repairexperiments. Therefore, for any one series of
12、experiments thesevariables (including controls) should be kept constant.3.1.4 Anesthetize the rat by intraperitoneal injection with a50-g/mL solution of sodium phenabarbitol (0.2 mL per 100 gbody weight) 10 min prior to the perfusion procedure. Otherproven anesthetics are also acceptable. Make sure
13、that theanimal is completely anesthetized, so that it feels no pain.3.1.5 Wet the abdomen thoroughly with 70 % ethanol andwipe with gauze for cleanliness to discourage loose fur fromgetting on the liver when the animal is opened.3.1.6 Make a V-shaped incision through both skin andmuscle from the cen
14、ter of the lower abdomen to the lateralaspects of the rib cage. Do not puncture the diaphragm or cutthe liver. Fold the skin and attached muscle back over the chestto reveal the abdominal cavity.3.1.7 Place a tube approximately 1 cm in diameter under theback to make the portal vein more accessible.3
15、.1.8 Move the intestines gently out to the right to reveal theportal vein. Adjust the tube under the animal so that the portalvein is horizontal.3.1.9 Put a suture in place (but not tightened) in the centerof the portal vein and another around the vena cava just abovethe right renal branch.1This pra
16、ctice is under the jurisdiction of ASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Sept. 10, 2003. Published September 2003. Originallyapproved in 1991. Last previous edit
17、ion approved in 1998 as E 1397 91 (1998).2The boldface numbers in parentheses refer to the list of references at the end ofthis practice.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.10 Perform perfusions with a peristaltic pum
18、p, thetubing of which is sterilized by circulation of 70 % ethanolfollowed by sterile water. Place a valve in the line so that theoperator may switch from the EGTA solution (see Annex A1)without disrupting the flow. Keep solutions at a temperaturethat results in a 37C temperature at the hepatic port
19、al vein.3.1.11 A peristaltic pump with a changeable pump head andsilicone tubing is suitable for performing the perfusion.3.1.12 Begin the flow of the 37C EGTA solution (seeAnnex A1) at 8 mL/min, and run to waste.3.1.13 Cannulate the portal vein with a 20 GA 114-in.catheter about 3 mm below the sutu
20、re. Remove the innerneedle and insert the plastic catheter further to about13 thelength of the vein and tie in place by the suture. Blood shouldemerge from the catheter. Insert the tube with the flowingEGTA (see Annex A1) in the catheter (avoid bubbles) and tapein place.3.1.14 Immediately cut the ve
21、na 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 tan color. If alllobes do not clear uniformly, the catheter has probably beeninserted too far into the portal vein.3.1.15 Tighten the suture around the vena cava a
22、nd increasethe flow to 20 mL/min for 2 min. The liver should swell at thispoint. In some cases gentle massaging of the liver or adjustingthe 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 i
23、n the liver.3.1.16 Switch the flow to the 37C collagenase solution for12 min. During this period, cover the liver with sterile 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 ens
24、ured that it willfunction properly.3.1.17 Allow the perfusate to flow onto the paper and collectby suction into a vessel connected by means of a trap to thevacuum line.3.1.18 After the perfusion is over, remove the catheter andgauze. Carefully remove the liver by cutting away the mem-branes connecti
25、ng it to the stomach and lower esophagus,cutting away the diaphragm, and cutting any remaining attach-ments to veins or tissues in the abdomen.3.1.19 Hold the liver by the small piece of attached dia-phragm and rinse with sterile saline or WEI (see Annex A1).3.1.20 Place the liver in a sterile petri
26、 dish and take to asterile hood to prepare the cells.3.2 Preparation of Hepatocyte Cultures:3.2.1 Place the perfused liver in a 60-mm petri dish andrinse with 37C WEI (see Annex A1). Remove extraneoustissues (fat, muscle, and so forth).3.2.2 Place the liver in a clean petri dish and add 30 mL offres
27、h collagenase solution (see Annex A1) at 37C.3.2.3 Carefully make several incisions in the capsule of eachlobe of the liver. Large rips in the capsule lead to largeunusable clumps of hepatocytes.3.2.4 Gently comb out the cells, constantly swirling the liverwhile combing. A sterile metal dog-grooming
28、 comb with teethspaced from 1 to 3 mm apart, or a hog bristle brush works well.3.2.5 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 (see Annex A1) using a wide-
29、boresterile pipet. Some laboratories report successful hepatocytepreparations when 3.2.1-3.2.8 are conducted with media atroom temperature or heated to 37C.3.2.6 Allow the cells to settle on ice for 5 to 10 min until adistinct interface is seen. Carefully remove and discard thesupernatant by suction
30、.3.2.7 Bring the cells to 50 mL with cold WEI (see AnnexA1). Resuspend the cells by pipeting with a wide-bore pipet.Gently pipet the suspension through a 4-ply layer of sterilegauze into a sterile 50-mL centrifuge tube.3.2.8 Centrifuge the cells at 50 times gravity for 5 min anddiscard the supernata
31、nt. Gently resuspend the pellet in ice-coldWEI (see Annex A1) with a wide-bore pipet.3.2.9 Some laboratories prefer to keep the cells on ice untilready for use, while others keep them at room temperature.Cells should be used as soon as possible, preferably within 1 h.3.2.10 Determine viability and c
32、ell 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
33、is an active process. Thus, if asufficient number of cells attach to conduct the experiment, itis a further indication of the viability of the culture.3.2.11 Place a 25-mm round plastic coverslip into each wellof 6-well culture dishes. 10.5 by 22-mm plastic coverslips and26 by 33-mm eight-chamber cu
34、lture dishes can also be used.Be sure to keep the proper side up as marked on the package.Add 4 mL of WEC (see Annex A1) to each well. Hepatocyteswill not attach to glass unless the slides have been boiled. Theuse of collagen-coated thermanox coverslips improves cellattachment and morphology.3.2.12
35、These procedures yield preparations consisting pri-marily of hepatocytes. Approximately 400 000 viable cells areseeded into each well and distributed over the coverslip byshaking or stirring gently with a plastic 1-mL pipet. Glasspipettes can scratch the coverslips.3.2.13 Incubate the cultures for 9
36、0 to 120 min in a 37Cincubator with 5 % CO2and 95 % relative humidity, to allowthe cells to attach.3.3 Labeling the Cultures:3.3.1 After the attachment period, wash the cultures oncewith 4 mL WEI (see Annex A1) per well to remove unattachedcells and debris. This is done by tilting the culture slight
37、ly,aspirating the media, and adding the fresh media at 37C. Becareful not to direct the stream from the pipet directly onto thecells.3.3.2 Prepare chemical solutions in3H-thymidine solution(WEI containing 10 Ci/mL3H-thymidine) (see Annex A1).Serial dilutions are generally employed. If employed, solv
38、entsfor the test substance, such as dimethyl sulfoxide (DMSO) orethanol, should not exceed a 1 % final concentration. Mostinvestigators try to limit the DMSO concentration to at orbelow 0.5 %, because of borderline toxic effects on someE 1397 91 (2003)2hepatocyte cultures at DMSO concentrations of 1
39、 %. Bothmedium alone and solvent controls should be included in theexperimental design. Concentrations of the test substanceshould be chosen that go just beyond cytotoxicity to about1000-fold below the cytotoxic concentration. Cytotoxicity canbe determined by trypan blue dye exclusion or lactic dehy
40、dro-genase (LDH) release of the cultures or by morphologicalexamination of the fixed and stained cells at the end of theexperiment. Typical concentration ranges are from 10 to 0.001mM. Relatively insoluble substances should be tested up totheir limit of solubility. Freely soluble, nontoxic chemicals
41、should not be tested at concentrations beyond 10 mM. A doseof 10 mM dimethylnitrosamine (DMN) is required to producea strong DNA repair response in the assay. In contrast,1,6-dinitropyrene induces DNA repair at concentrations as lowas 0.00005 mM.3.3.3 Remove the WEI (see Annex A1) and replace with 2
42、mL of3H-thymidine solution (see Annex A1) containing thedissolved test chemical. Place the cultures in the incubator for16 to 24 h. During this period the compound may be metabo-lized. If DNA damage occurs, it will be repaired, resulting inincorporation of the3H-thymidine (see Annex A1).3.3.4 Wash c
43、ultures twice with 4 mL WEI (see Annex A1)per well.3.3.5 The remainder of these procedures are done withsolutions at room temperature. Replace the medium with 4 mLof a 1 % sodium citrate solution and allow the cultures to standfor 10 min to swell the nuclei. The purpose for swelling thecells is that
44、 the larger nuclei are more easily scored than theunswollen nuclei where the silver grains are more bunchedtogether. Some operators prefer to omit this step. There is noevidence that swelling the nuclei yields any significant differ-ence in the results compared to when the nuclei are notswollen.3.3.
45、6 Replace the sodium citrate solution with 3 mL of a 1to 3 ratio of acetic acid to absolute ethanol solution for 10 minto fix the cells. Repeat this twice more for a total fixing time ofat least 30 min.3.3.7 Wash wells 2 to 6 times each, with deionized distilledwater.3.3.8 Remove coverslips from the
46、 wells and place cell-side-up on the edge of the dish covers to dry in a dust-freelocation at room temperature.3.3.9 When dry, mount coverslips cell-side-up on micro-scope slides with mounting compound. Coverslips should bemounted about 1 cm from the unfrosted end of the slide. Giveeach slide a uniq
47、ue identifying number.3.3.10 At this point, the cultures can be examined for grosscytotoxicity. If chemical treatment at the higher doses hasresulted in there being no cells on the slides, they need not besubjected to autoradiography.3.4 Autoradiography:3.4.1 Any proven autoradiographic technique th
48、at yieldssilver grains in proportion to the amount of incorporatedlabeled thymidine may be used. Presented in 3.4.2-3.4.14 is atypical procedure.3.4.2 All steps involving photographic emulsions should bedone in total darkness. If absolutely necessary a red safelightfilter may be used sparingly.3.4.3
49、 Mount slides for each dose in plastic slide grips.Duplicate slides may be held in reserve.3.4.4 Mount a 50-mL disposable plastic beaker with tapeinto a slightly larger jar full of water. Place this assembly intoa 42C water bath and allow to reach the bath temperature.3.4.5 Kodak NTB-23emulsion is most commonly used. Theemulsion may be used undiluted or can be diluted toa1to1ratio with distilled water. If the emulsion is diluted, take care touse double distilled or ultrapure water; thoroughly mix thesolution but avoid formation of air bubbles. Undiluted emul-sion saves a st
