Consensus statement on prevention and management of adverse effects following rejuvenation procedures with hyaluronic acid-based fillers.

Facial fillers play an important role in the correction of facial changes associated with ageing. They offer quick treatments in the outpatient setting with minimal subsequent downtime that provide predictable, natural-looking, long-lasting results. Adverse reactions after hyaluronic acid injections tend to be mild or moderate and rather temporary. However, as with all injected or implanted biomaterials, severe adverse events can occur and patients must be fully informed of potential risks prior to undergoing treatment. A panel of experts from Germany (D), Austria (A) and Switzerland (CH) developed recommendations, and this study provides the 'DACH Consensus Recommendations' from this group specifically on the use of hyaluronic acid fillers. The aim is to help clinicians recognize potential risks and to provide guidance on how best to treat adverse events if they arise. Contraindications to hyaluronic acid fillers are also detailed, and ways to prevent adverse events occurring are discussed. Hyaluronic acid-based products are claimed to be very close to an ideal tissue augmentation agent; nevertheless, profound medical, anatomical and product knowledge are of paramount importance to minimize the occurrence of adverse reactions.

[Lidocaine-containing hyaluronic acid filler on a CPM® basis for lip augmentation : Experience from clinical practice]. / Lidocainhaltiger Hyaluronfiller auf CPM®-Basis zur Lippenaugmentation : Erfahrungsberichte aus der Praxis.

BACKGROUND: Lip augmentation with hyaluronic acid fillers is an established procedure. As monophasic polydensified hyaluronic acid products with variable density CPM-HAL1 (Belotero® Balance Lidocaine) and CPM-HAL2 (Belotero® Intense Lidocaine) are qualified for beautification and particularly natural-looking rejuvenation, respectively. OBJECTIVES: Assessment of handling and outcome of lip augmentation using the lidocaine-containing hyaluronic acid fillers CPM-HAL1 and CPM-HAL2. MATERIALS AND METHODS: Data from patients who received lip augmentation by means of bautification and/or rejuvenation using CPM-HAL1 and/or CPM-HAL2 were documented. Observation period was 4 months, with assessment of natural outcome, evenness, handling, fluidity, distribution, malleability, tolerability, as well as patient satisfaction and pain. RESULTS: In total, 146 patients from 21 German centres participated. Physicians rated natural outcome and evenness as good or very good for > 95 % of patients. Handling, fluidity, distribution and malleability were assessed for both fillers as good or very good in > 91 % of patients. At every evaluation point, more than 93 % of patients were very or very much satisfied with the product. A total of 125 patients (85.6 %) experienced transient injection-related side effects. Pain intensity during the procedure was mild (2.72 ± 1.72 on the 0-10 pain assessment scale) and abated markedly within 30 min (0.42 ± 0.57). CONCLUSIONS: Lip augmentation with hyaluronic acid fillers produced a long-term cosmetic result. Due to the lidocaine content, procedural pain was low and transient. Accordingly, a high degree of patient satisfaction was achieved that was maintained throughout the observation period.

[Body contouring]. / Körperkonturierung.

In our contemporary postmodern society, a modified perception of the human body is accompanied by an increasing demand for body shaping procedures. The treatment needs to be effective but it is just as important that they are safe and can be easily integrated into the daily working and routine schedule. While the options for minimally invasive volume addition are largely limited to injectable implants based on hyaluronic acid or autologous fat, a multitude of options are available for volume reduction. Before deciding on the method of choice, the following needs to be considered: which indications need to be treated, the extent of the reduction in volume and how much pain and possible undesired reactions the patient is prepared to accept.

[Complications of liposuction]. / Komplikationen bei Eingriffen am Fettgewebe.

Liposuction is the most frequent aesthetic procedure worldwide for adipose tissue reduction and treatment of lipedema. It is being employed with increasing frequency. In 2010, in the USA more than 200.000 liposuctions were performed. Apart from aesthetic indications, liposuction also is suitable for treatment of benign adipose tissue diseases. This intervention is not a simple procedure but requires extensive knowledge and experience to prevent irreversible medical or aesthetic complications. Severe complications including necrotizing fasciitis, toxic shock syndrome, hemorrhage, perforation of inner organs und pulmonary embolism - some even with lethal outcome - occasionally have been reported. These complications were mostly due to inadequate hygiene measures, inappropriate patient selection, use of excessive local anesthesia during mega-liposuction (tumescent technique) and inadequate post-operative surveillance. The complication rate usually reflects a lack of medical experience as well as technical inadequacies.

Cytochrome P-450 induction by phenobarbital and 3-methylcholanthrene in primary cultures of hepatocytes.

The characteristic hepatocellular changes resulting from phenobarbital administration in vivo, namely an increase in the levels of cytochrome P-450 and proliferation of membranes of the smooth endoplasmic reticulum, have been demonstrated in primary cultures of nonreplicating hepatocytes on floating collagen membranes. Addition of methylcholanthrene to the medium resulted in an increase in cytochrome P-448 within 48 hours, whereas the phenobarbital induction of P-450 required 5 days. These results demonstrate that responses induced in adult liver cells in vivo by phenobarbital can be reporoduced in cultured hepatocytes, contrary to previous reports.

Hormonal regulation and the effects of glucose on tyrosine aminotransferase activity in adult rat hepatocytes cultured on floating collagen membranes.

Adult rat parenchymal hepatocytes can be maintained in primary culture on floating collagen membranes of prolonged periods of time. In this system the enzyme tyrosine aminotransferase is induced by glucagon, (10(-6) to 10(-8) M) hydrocortisone (10(-5) to 10(-8) M), and cyclic adenosine 3':5'-monophosphate (cAMP) (10(-4) to 10(-5) M). Epinephrine (10(-4) M) induces the enzyme only in the presence of hydrocortisone. Addition of actinomycin D inhibited the induction of tyrosine aminotransferase by hydrocortisone and cAMP. Maintenance of the cultured hepatocytes in the presence of glucose (3g/liter) results in partial suppression of the inducing effects of glucagon and cAMP. Cyclic quanosine 3':5'-monophosphate does not mimic the effects of glucose. These results demonstrate that the phenomenon of glucose repression of enzyme induction, demonstrated in vivo in mammalian liver, is independent of changes in levels of serum hormones, which occur in vivo as a result of glucose administration. This study also demonstrates that glucose repression is not mediated by changes in intracellular levels of cAMP and cyclic quanosine 3':5'-monophosphate.

Use of primary cultures of adult rat hepatocytes on collagen gel-nylon mesh to evaluate carcinogen-induced unscheduled DNA synthesis.

The procarcinogen, 2-acetylaminofluorene, the direct-acting carcinogen, methyl methanesulfonate, and two other hepatocarcinogens, thioacetamide and urethan, were tested for their ability to elicit unscheduled DNA synthesis in adult rat hepatocytes maintained in primary culture on collagen gel-nylon mesh. The carcinogens, dissolved in dimethyl sulfoxide were added to 6-hr or to 28-hr cultures along with [methyl-3H]thymidine (1 muCi/ml medium) in the presence of 10 mM hydroxyurea. Twelve hr later, the hepatocytes were harvested from the cultures with collagenase, and their DNA was purified on CsCl isopyknic gradients. Unscheduled DNA synthesis was measured as the increase in [methyl-3H]thymidine radioactivity incorporated per microgram DNA of the carcinogen-treated cultures as compared with that of control cultures. Both 2-acetylaminofluorene and methyl methanesulfonate demonstrated a concentration-dependent stimulation of unscheduled DNA synthesis in the 6-hr hepatocyte cultures. However, the response of the 28-hr cultures to these two carcinogens was absent unless the hepatocytes were preincubated for 22 hr in culture medium supplemented with 10(-5) M dexamethasone and 10(-6) M glucagon or in a more complete hormone-supplemented medium. Thioacetamide and urethan, on the other hand, failed to elicit a concentration-dependent unscheduled DNA synthesis under these conditions. The results obtained with this culture system are similar to those of other short-term tests for chemical carcinogenicity and support the potential use of the collagen gel-nylon mesh-hepatocyte primary culture as an in vitro screen for chemical carcinogens. Furthermore, this study suggests the importance of specific hormones in maintaining the capability for repair of DNA damage produced by carcinogenic and mutagenic chemicals in cultured hepatocytes.

Effect of age on the formation of small-cell colonies in cultures of primary rat hepatocytes.

The proliferation of primary cultured rat hepatocytes was observed in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml of epidermal growth factor. These proliferating cells were mainly mononucleate and formed small-cell colonies after Day 4. The small cells in focal colonies were surrounded by typical hepatocytes and were stained immunocytochemically with anti-rat albumin and anti-cytokeratin 8 antibodies. This suggests that the cells in the small-cell colonies were derived from hepatocytes. The frequency of appearance of small-cell colonies with age was examined by the use of primary cultured hepatocytes isolated from the livers of rats between the ages of 3 wk and 90 wk. In the cells from 4- to 5-wk-old rats, about 58 colonies per 1000 attached cells appeared 144 h after plating; the number of colonies rapidly decreased to about 25 in 6- to 8-wk-old rats. In adult rats, about 17 colonies were seen, and only about five colonies were observed in rats more than 80 wk old.

Ultrastructure of adult rat hepatocytes cultured on floating collagen membranes.

The ultrastructure of primary hepatocytes cultured for 2 to 17 days on floating collagen membrane was evaluated. A pellet of the hepatic cell suspension used to inoculate the collagen membranes contained some single cells and many aggregates of two, three, or four cells. Desmosomes were split during the perfusion, but tight junctions and gap junctions remained intact. By 2 days in culture, the hepatocytes had formed a monolayer of cells of polygonal shape with newly synthesized desmosomes between cells. Since the flexible floating collagen membrane decreases in size as the monolayer forms, the hepatocytes do not flatten out, as is characteristic of cells cultured on a rigid substrate. Hepatocytes in culture for 10 days or less exhibited large lamellar arrays of rough endoplasmic reticulum, well developed Golgi complexes, and structures resembling bile canaliculi, which possess tight junctions and desmosomes separating them from the intercellular space. Microfilaments oriented parallel to the plasma membranes of adjoining cells and in an intermeshed network at the edge of the monolayer and beneath the plasma membrane bordering the medium increased in size and number in older cultures. After 17 days in culture, the cells maintained tight junctions, desmosomes, Golgi complexes, and rough endoplasmic reticulum in small lamellar stacks and in small vesicles. Since hepatocytes on the floating collagen membrane retain most of the subcellular structural elements characteristic of normally functioning hepatocytes for 2.5 weeks, this system may be valuable for future experiments involving drug metabolism and carcinogenesis in vitro.

Unscheduled DNA synthesis induced by procarcinogens in suspensions and primary cultures of hepatocytes on collagen membranes.

Unscheduled DNA synthesis was induced by procarcinogens in freshly isolated suspensions and primary cultures (6 days old) of hepatocytes on collagen membranes. Incorporation of [3H]thymidine in the presence of hydroxyurea was used to measure unscheduled DNA synthesis. When hepatocellular DNA was isolated on cesium chloride gradients, significant levels of unscheduled DNA synthesis were measured. Similar concentrations of procarcinogens elicited higher levels of unscheduled DNA synthesis in hepatocellular suspensions than in primary cultures. The results demonstrate that hepatocytes cultured on collagen membranes can metabolize chemical carcinogens. Suspensions of freshly isolated hepatocytes, however, are more active in procarcinogen metabolism than are those of primary cultures. The selective advantages of the two systems of hepatocytes can be utilized for the establishment of short-term in vitro screening systems of mutagens and carcinogens.

Specific chromosomal changes in albumin simian virus 40 T antigen transgenic rat liver neoplasms.

Hepatocytes isolated from 3-month-old female rats bearing the albumin promoter/enhancer SV40 T antigen construct as a transgene demonstrated a 20% aneuploidy rate and a significant duplication of chromosome 1. Other chromosome changes were observed but were not statistically significant. At this time in the development of hepatic lesions, only a relatively small number of microscopic altered hepatic foci could be noted. By contrast, hepatocytes isolated from the age-matched nontransgenic controls demonstrated only 1% aneuploidy. One hundred % of the metaphase spreads isolated from hepatocellular neoplasms in transgenic rats were aneuploid. Although there were many random changes, 70% of the neoplastic cells demonstrated an amplification of all or portions of chromosome 1q. Only 2% of the neoplastic cells had both a trisomy and a duplication. The smallest region of chromosome 1 that was duplicated was that between bands q3.7 and q4.3. A loss of chromosome 3 was detected in 50% of the neoplasms, as well as a loss of chromosome 6 in 72% of the neoplastic cells. The carcinomas with the highest proliferation rate had also lost at least one copy of chromosome 15 in 70% of the cells. The loss of chromosomes 3, 6, and 15 indicates that these regions may harbor one or more tumor suppressor genes. The amplification of a specific region of chromosome 1 is thus the first karyotypic alteration that can be identified in hepatocytes from livers from which hepatic neoplasms will arise. This indicates that expression or repression of one or more genes in this region may confer a growth advantage to preneoplastic hepatocytes, facilitating their transit to the neoplastic state in the stage of progression. Changes in chromosomes 3, 6, and 15 that occur subsequent to duplication of the q3.7-q4.3 region of chromosome 1 are changes possibly reflecting alteration of tumor suppressor genes with further enhancement of neoplastic growth.

Karyotypic changes in a multistage model of chemical hepatocarcinogenesis in the rat.

Karyotypic analysis of the stages of rat hepatocarcinogenesis has been facilitated by the development of an initiation-promotion-progression (IPP) protocol that permits separation and characterization of morphologically normal and altered hepatocytes in each of these three stages. The expression of the membrane antigen gamma-glutamyl transpeptidase (GGT) during the promotion and progression stages of rat hepatocarcinogenesis permits the isolation, culture, analysis, and comparison of hepatocytes in the two stages, which express this marker of carcinogenesis. Female rats were administered 10 mg diethylnitrosamine/kg at 5 days of age. One group of initiated rats was maintained on dietary phenobarbital admixed into a laboratory chow diet at 0.05% for 9 months after weaning (promotion protocol). This initiation-promotion (IP) group was compared with one subjected to the complete IPP protocol. The IPP group was initiated with diethylnitrosamine, maintained on phenobarbital for 6 months after weaning, and then subjected to a 70% partial hepatectomy and administered 100 mg ethylnitrosourea/kg 24 h later. These rats on the IPP protocol were then maintained on phenobarbital for an additional 3 months prior to sacrifice. At sacrifice, single hepatocyte suspensions were obtained and separated into populations of cells expressing or not expressing GGT. These hepatocyte populations were cultured separately and subjected to standard cytogenetic analysis. At least five animals per treatment and 100 metaphase spreads of good morphology per animal were examined. Although GGT- cells from the IP protocol were 80% tetraploid and 20% diploid, the GGT+ hepatocytes were greater than 90% diploid. The GGT+ cells from this protocol had a low rate of random aneuploidy (4.0 +/- 1.3%) compared with corresponding cells from the IPP protocol, but a higher level of background aneuploidy compared with GGT- cells from the IP protocol. The GGT+ hepatocytes from animals on the IPP protocol had a 35% incidence of aneuploidy. In addition, the GGT+ population had a 28 +/- 5% incidence of chromosomal breakage and a 17 +/- 5% incidence of chromosomal rearrangements. The primary nonrandom chromosomal changes observed in cells from the IPP protocol included duplication of all or part (1q37-43) of chromosome 1 and the loss of chromosomes 3p and/or 6q. These studies indicate that rat hepatocytes in the stage of promotion are euploid, whereas those in the stage of progression exhibit considerable genetic instability. The presence of multiple copies of chromosome 1 or a duplication of a region of this chromosome indicates that alteration of gene dosage for one or more of the genes present in this region is critical to the neoplastic conversion of rat hepatocytes, whereas the loss of all of 3p and the last light band of 6q may indicate the presence of tumor suppressor genes. Thus, the IP and IPP protocols coupled with the ability to isolate GGT+ and GGT- hepatocytes permit the differential cytogenetic characterization of the stages of promotion and progression in rat hepatocarcinogenesis.

Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens.

Technical modifications of the quantitative determination of unscheduled DNA synthesis in cultured hepatocytes are described which allow for the rapid identification of potentially carcinogenic chemicals on a large-scale screening basis. The test is based on the biochemical quantification of [methyl-3H]thymidine incorporation into DNA in the presence of hydroxyurea following isolation of nuclei from hepatocytes treated with the agent under study. This procedure ("nuclei procedure") eliminates most of the background radioactivity which otherwise obscures the stimulation of DNA repair synthesis by agents that induce a relatively weak response. By combining the nuclei procedure with a double-labeling technique, test results can be obtained within a few hr after exposure of hepatocytes to the test agents. A test series involving 41 agents confirmed the reliability of the nuclei procedure for the assay of DNA repair synthesis. In addition, chemicals which had yielded conflicting results previously in the autoradiographic hepatocyte DNA repair test, such as 4-acetylaminofluorene, or which had passed unrecognized in previous in vitro tests, such as the potent liver carcinogen methapyrilene hydrochloride, scored clearly positive in our test protocol.

Tamoxifen induces hepatic aneuploidy and mitotic spindle disruption after a single in vivo administration to female Sprague-Dawley rats.

Tamoxifen has found extensive use in the treatment of all stages of human breast cancer. The efficacy of tamoxifen treatment for the prevention of second primary tumors and its chemosuppressive action in animal models have led to initiation of clinical trials to test its efficacy for prevention of this disease in women. Recently, tamoxifen has been shown to induce hepatocellular carcinomas in rats. For determination of the mechanism of induction of these tumors and assessment of the possibility of risk of human cancer development from tamoxifen treatment, female Sprague-Dawley rats (five rats per treatment) were administered tamoxifen at doses ranging from 0.3 to 35 mg/kg. One day after treatment, the rats were sacrificed, and the hepatocytes were isolated and cultured for 50 h. Colcemid was added 3 h prior to harvest, and the hepatocytes were then prepared for karyotypic evaluation. One hundred metaphase spreads were examined per animal. Tamoxifen treatment resulted in the induction of aneuploidy in approximately 70% of the examined hepatocytes at the doses used. In addition, premature condensation (2-10%) and endoreduplication (5-10%) were observed in hepatocytes of rats treated with tamoxifen. Furthermore, exchanges between chromosomes as well as chromosome breakage were observed. Examination of the cultured hepatocytes from rats treated with tamoxifen by electron microscopy demonstrated both unipolar spindles and incompletely elongated spindles. Exposure of rats to a single in vivo dose of tamoxifen produced multiple changes in rat hepatocytes including clastogenic damage at doses comparable to that administered to humans. The occurrence of aneuploidy induction, premature condensation, chromosome breakage, and improper mitotic spindle formation indicates that risk versus benefit of tamoxifen treatment should be carefully evaluated.

Ploidy and specific karyotypic changes during promotion with phenobarbital, 2,5,2',5'-tetrachlorobiphenyl, and/or 3,4,3'4'-tetrachlorobiphenyl in rat liver.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.

Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers.

Altered hepatic foci (AHF) were analyzed by quantitative stereology on frozen serial sections stained sequentially for gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphate (ATPase), glucose-6-phosphatase (G6Pase), and the placental isoenzyme of glutathione S-transferase (GST). Livers for these analyses were obtained from both male and female rats of different ages which had been subjected to initiation with a nonnecrogenic dose of diethylnitrosamine following a 70% partial hepatectomy with subsequent phenobarbital (PB) feeding. Different combinations of these four marker alterations (from single marker to four-marker combinations) were used to analyze the data, and the results were compared for their ability to detect AHF. In rats on the above protocol, GST was the single most effective marker, exhibiting a high sensitivity for scoring both number and volume of foci. There was a high degree of overlap with GGT. The combination of the four different markers, GST/GGT/ATPase/G6Pase, scored 80% more foci in number and 60% more in volume than the routinely used GGT/ATPase/G6Pase method. When all four markers were used to score AHF, PB promotion was equally effective in both sexes at weaning and at 6 months of age, but at 1 year of age males showed a dramatic reduction in the effectiveness of PB as a promoting agent, both for number and volume percentage of liver occupied by AHF. On the other hand, initiation was more effective in the male at weaning and at 6 months of age, although by the 12-month point no distinction between the sexes could be made. When only GGT was used as a marker, promotion by PB appeared to be markedly less effective in males than in females at all ages. In the absence of PB administration, both the number and volume fraction of AHF in the livers of both males and female increased with age. Likewise, both the number of AHF per liver and their volume fractions increased with age in both sexes when uninitiated animals were fed PB, although only after a 6-month lag in females. These experiments demonstrate that the stages of initiation and promotion in hepatocarcinogenesis in the rat as monitored by the number and volume percentage occupied of AHF are altered by both the age and the sex of the animal. The combination of GGT and GST identified all AHF scored by the GST/GGT/ATPase/G6Pase set of markers and thus may be the most efficient combination of markers of AHF resulting from promotion by PB.

[Current Perceptions of Lipofilling on the Basis of the New Guideline on "Autologous Fat Grafting"]. / Aktuelle Erkenntnisse zur Eigenfett Transplantation anhand der neuen Leitlinie "Autologe Fetttransplantation".

Introduction: Autologous fat transfer has recently become an increasingly popular surgical procedure and comprises harvesting, processing and transplantation of adipose tissue, as well as professional follow-up care. This method, as a surgical procedure, can be utilised for trauma-, disease- or age-related soft tissue volume deficits and soft tissue augmentation. As usage is increasing, but the variables of fat harvest, specific indications and fashion of fat transfer are poorly defined, there is a great demand for development of a guideline in the field of reconstructive and aesthetic surgery. Methods: All relevant points were discussed within the scope of a consensus conference including a nominal group process of all societies involved in the procedure and ratified with a strong consensus (>95%). Literature from the standard medical databases over the last 10 years was retrieved, studied and specific guidelines were concluded. Results: Consensus was achieved among all professionals involved on the following points: 1. definition 2. indication/contraindication, 3. preoperative measures 4. donor sites 5. techniques of processing 6. transplantation 7. follow-up care 8. storage 9. efficacy 10. documentation 11. evaluation of patient safety. Conclusion: Definite indications and professional expertise are paramount for autologous fat tissue transfer. Successful transfers are based on the use of correct methods as well as specific instruments and materials. Autologous adipose tissue transplantation is considered to be a safe procedure in reconstructive and aesthetic surgery, due to the low rate of postoperative complications and sequelae.

Effect of hypolipidemic peroxisome proliferators on unscheduled DNA synthesis in cultured hepatocytes and on mutagenesis in Salmonella.

The peroxisome proliferators Wy-14,643, BR-931, nafenopin and ciprofibrate were tested in the primary hepatocyte culture-unscheduled DNA synthesis assay and in the Ames Salmonella microsome mutagenicity assay. The amount of unscheduled DNA synthesis (UDS) in hepatocytes was determined by quantifying the amount of [3H]thymidine incorporated into DNA in the presence of hydroxyurea after isolation of nuclei from hepatocytes treated with the test agent. Wy-14,643 and BR-931 induced unscheduled DNA synthesis in rat hepatocytes, whereas nafenopin and ciprofibrate had no effect. All of the peroxisome proliferators were negative in the Ames Salmonella assay.

Aging lowers steady-state antioxidant enzyme and stress protein expression in primary hepatocytes.

It has been reported that the isolation and culture of primary hepatocytes can compromise cellular ability to constituitively express antioxidant enzyme (AE) genes, making it difficult to study their regulation ex vivo. In the present study, the steady-state expression of manganese-containing superoxide dismutase, copper- and zinc-containing superoxide dismutase, catalase, and glutathione peroxidase was assessed in primary hepatocytes isolated from young and senescent rats and cultured in MATRIGEL: There was no change in steady-state superoxide dismutase protein or activity levels in cells collected from young animals and cultured for 7 days. Catalase expression was initially increased, and then it declined 30%. In contrast, superoxide dismutase expression declined 60% and catalase expression declined 50% in cells from senescent animals. Constitutive and inducible 70-kDa heat shock protein expression increased coincident with declining AE levels in the young cells but not senescent cells. For both age groups, electron micrographs showed rounded hepatocytes with abundant rough endoplasmic reticulum, mitochondria, and peroxisomes. Hepatocytes were organized into clusters of 6-12 cells surrounding a large central lumen devoid of microvilli. Each cluster also contained smaller microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes. Gap junctions in the plasma membrane suggest that hepatocytes were capable of intercellular communication. We conclude that the Matrigel system can be used to study AE regulation in primary hepatocytes from young and senescent animals, provided that experiments can be conducted within a time frame of 5-7 days in culture. These data also support the hypothesis that aging compromises hepatocellular ability to maintain AE status and upregulate stress protein expression.