Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: interindividual variation to carcinogen exposure.

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7, 12-dimethylbenz(a) anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 micrometer NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [3H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 micrometer DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure or mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 micrometer NA-AAF dose, as estimated by [3H] thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

DNA repair and replication in human fibroblasts treated with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

DNA repair and replication were examined in diploid human fibroblasts after treatment with (±)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I). Unscheduled DNA synthesis exhibited a linear response to BPDE-I concentrations up to 1.5 μM and a saturation plateau after higher concentrations. Maximal unscheduled DNA synthesis was observed in the first hour after treatment with synthesis diminishing progressively thereafter. Half-maximal unscheduled DNA synthesis was seen within 4–6 h after treatment with 0.7 μM BPDE-I. DNA replication was inhibited by BPDE-I in a dose- and time-dependent fashion. The mechanisms of this inhibition were characterized by velocity sedimentation of pulse-labeled nascent DNA in alkaline sucrose gradients. Very low concentrations of BPDE-I (0.03 and 0.07 μM) were found to inhibit replicon initiation by up to 50% within 30–60 min after treatment. Recovery of initiation following these low concentrations was evident within 3 h after treatment. Higher concentrations of carcinogen inhibited DNA synthesis in active replicons. This effect was manifested by a reduction in incorporation of precursor into replication intermediates of greater than 1·10⁷ Da with the concurrent production of abnormally small nascent DNA. When viewed 45 min after treatment with 0.17 μM BPDE-I the combination of these two effects partially masked the inhibition of replicon initiation. However, even after treatment with 0.33 μM BPDE-I an effect on initiation was evident. These results reveal a pattern of response to BPDE-I that is quite similar to that produced by 254 nm radiation.     

Biomonitoring of genotoxic exposure among stainless steel welders.

A biosurvey in the Danish metal industry measured the genotoxic exposure from stainless steel welding. The study comprised measurements of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), unscheduled DNA synthesis (UDS) in peripheral lymphocytes and serum immunoglobulin G. Environmental monitoring of welding fumes and selected metal oxides, biomonitoring of chromium and nickel in serum and urine and mutagenic activity in urine, and evaluation of semen quality were also done. Manual metal arc (MMA) welding and tungsten inert gas (TIG) welding were the dominant welding processes. A higher frequency of chromosomal aberrations, classified as translocations, double minutes, exchanges and rings, was observed in stainless steel welders than in non-welders. SCE was lower in welders working with both MMA and TIG welding than in reference persons. N-Acetoxy-N-acetylaminofluorene (NA-AAF)-induced UDS was lower in 23 never-smoking welders than in 19 unexposed never-smokers. Smoking was a confounding factor resulting in significantly higher CA, SCE, NA-AAF binding to DNA and mutagenic activity in urine. Age was also a confounder: CA, SCE, NA-AAF binding to DNA and UDS increased significantly with age. No significant correlation between SCE and CA or between CA and UDS was found. UDS decreased significantly with increasing lymphocyte count and a higher lymphocyte count was seen in MMA welders than in reference persons and in smokers than in non-smokers. Differences in the composition among lymphocytes in exposed persons compared with non-exposed are suggested. MMA welding gave the highest exposure to chromium, an increased number of chromosomal aberrations and a decrease in SCE when compared with TIG welding. Consequently improvements in the occupational practice of stainless steel welding with MMA is recommended.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Effects of nicotinamide on NAD and poly (ADP-ribose) metabolism in DNA-damaged human lymphocytes

The effect of nicotinamide on unscheduled DNA synthesis was studied in resting human lymphocytes. In cells treated with UV irradiation or with MNNG, nicotinamide caused a two-fold stimulation of unscheduled DNA synthesis and retarded the rate of NAD⁺ lowering caused by these treatments. Nicotinamide also reduced the burst of poly(ADP-ribose) synthesis caused by MNNG treat-ment. Thus under conditions that it enhances unscheduled DNA synthesis, nicotinamide causes marked effects on the metabolism of NAD⁺ and poly(ADP-ribose). The effect of nicotinamide on unscheduled DNA synthesis was shown to be independent of protein or polyamine synthesis.     

Sister-chromatid exchanges in human lymphocytes after a non-S-phase incubation period to allow excision DNA repair-in vitro exposure to N-acetoxy-2-acetylaminofluorene and ethylene oxide

Sister-chromatid exchanges (SCE) were analyzed in human peripheral blood lymphocytes at the baseline level, after induction of DNA damage by N-acetoxy-2-acetylaminofluorene (NA-AAF) and ethylene oxide (EO), and after a subsequent 18-h DNA-repair incubation period. There was a significant difference between the baseline SCE frequencies as compared to those after 1 h of NA-AAF or EO treatment. There was no significant difference between the SCE frequencies after 1 h of NA-AAF treatment and those after 18 h of DNA-repair incubation, suggesting that only a low level of NA-AAF damage to DNA had been removed. However, there was a significant difference between the SCE frequencies after 1 h of EO treatment and those after 18 h of DNA-repair incubation, indicating that a significant level of EO induced DNA lesions had been repaired. Thus, it seems likely that the EO induced DNA damage is more easily recognized, and hence more rapidly repaired than the NA-AAF induced damage. The reason for this may be the different chemical nature of the DNA lesions induced, which, in turn, leads to different kinetics of DNA repair.     

Influence of duration of exposure to styrene oxide on sister chromatid exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro

Styrene-7,8-oxide, an intermediate of styrene, is a known alkylating mutagen. The present study was carried out to investigate the influence of duration of exposure to styrene-7,8-oxide (styrene oxide) on induction of sister chromatid exchanges (SCEs) and inhibition of cell-cycle kinetics using cultured human blood lymphocytes in vitro. Phytohemagglutinin-stimulated whole-blood lymphocyte cultures obtained from heparinized whole blood from healthy donors were exposed to 100 μM styrene oxide for 22, 36, 48 and 72 h. A reduction of SCEs induction with increase in duration of exposure to styrene oxide was observed, i.e. a clear significant inverse relationship between exposure time and frequencies of SCEs induction due to styrene oxide was obtained. Styrene oxide induces significant elevations in unscheduled DNA synthesis DNA repair as well as S-phase synthesis in human blood lymphocytes in vitro, depending on the duration of exposure. The decrease in the induction of SCEs due to styrene oxide with increasing duration of its exposure may be principally due to an increased DNA repair and partly due to an increasing metabolic transformation to styrene glycol with increasing duration of its exposure as well as to some extent due to cell death at the maximum period of exposure, i.e. 72 h. Although the proliferations of lymphocytes exposed to 100 μM styrene oxide were significantly inhibited at different durations of exposure, no linear relationship between the replication index and the duration of exposure was noticed (r=0.47, p>0.05). Similarly, there was no relationship between replication index and SCE frequency (r=−0.36, p>0.05), suggesting that these two parameters may reflect two different endpoints for the cytogenotoxic effects of styrene oxide.     

Unscheduled DNA synthesis correlated to alkylation of hemoglobin in individuals occupationally exposed to propylene oxide

Exposure to propylene oxide was determined previously by the degree of alkylation of hemoglobin measured on the histidine residue as N-3-(2-hydroxypropyl) histidine, using blood samples from 8 propylene oxide-exposed employees and 13 unexposed referents. Mononuclear leukocytes isolated from the same blood samples were used to quantify DNA repair proficiency following an in vitro challenge with the carcinogen, N-acetoxy-2-acetylamino-fluorene. Decreases in the DNA repair proficiency index correlated significantly to in vivo exposure levels to propylene oxide (r = –0.64, p <0.03). These data suggest a possible short-term biological assay for monitoring the in vivo genotoxic effects of propylene oxide exposure in the human population.Abbreviations EO ethylene oxide - NA-AAF N-acetoxy-2-acetylaminofluorene - HOPrHIS N-3-(2-hydroxypropyl) histidine - PO propylene oxide - UDS unscheduled DNA synthesis     

Studies on the regulation of lymphocyte reactivity by normal and activated macrophages.

To determine the potential role of macrophages as regulators of the immune response, the effect of mouse peritoneal macrophages on transforming mouse spleen lymphocytes was investigated. Mitogen and antigen stimulated lymphocyte transformation, as measured by DNA synthesis, was enhanced by all concentrations of normal macrophages tested, but only by low concentrations of activated macrophages. High concentrations of activated macrophages markedly inhibited lymphocyte transformation. This inhibition occurred whether lymphocyte DNA synthesis was measured by incorporation of [³H]TdR or of ³²P. Activated macrophages cultured with lymphocytes within 4 hr of being removed from the peritoneal cavity inhibited lymphocyte transformation. When activated macrophages were cultured alone for 24 or more hours before addition of lymphocytes, enhancement of transformation was noted. Once lymphocytes were exposed to activated macrophages, they could not be induced to undergo transformation in the presence of Con A. Whereas heat-killed activated macrophages, which appeared intact morphologically, lost their capacity to inhibit lymphocyte transformation, macrophages treated with mitomycin C to inhibit DNA synthesis retained this capacity. Syngeneic and allogeneic macrophages had similar inhibitory ability. Supernatants from cultures of many cell types (including normal or activated macrophages, lymphocytes, lymphocytes plus macrophages, and L cells) inhibited [³H]TdR incorporation by both mitogen stimulated lymphocytes and tumor cells. These studies demonstrate the capacity of macrophages to regulate lymphocyte transformation in vitro and suggest a role for these cells as regulators of cell-mediated immunity in vivo.     

Quantification of unscheduled DNA synthesis in mononuclear leukocytes of the horse

This study compares the relationship between N-acetoxy-2-acetylaminofluorene (NA-AAF) and u.v. induced unscheduled DNA synthesis (UDS) and their respective relationships to age and blood pressure in horse mononuclear leukocytes with earlier, similar investigations on human leukocytes. U.v. induced UDS was found to proceed more rapidly than NA-AAF induced UDS. A pronounced lag period associated with the rapid demand for 3H-dThd into DNA after u.v. damage was observed. NA-AAF induced UDS correlated significantly with NA-AAF binding, age and the blood pressure of male horses. UDS values, induced by either method, were about half the level calculated for human leukocytes.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Regulation of repair of naturally occurring DNA strand breaks in lymphocytes

Mouse lymphocytes have been shown to contain DNA strand breaks that were repaired within 2h of onset of culture with mitogen. Inhibitors of ADP ribosylation prevented this repair and blocked cell proliferation. The mitogen concanavalin A caused the internal concentration of NAD⁺, the substrate of the ADP ribose polymerase, to rise to about double that of resting cells within 45 min of stimulation. Addition of 300 μm nicotinamide to the culture in absence of mitogen also resulted in a similar increase in internal [NAD⁺], resulting in increased ADP ribosylation activity (measured in permeabilized cells) and in joining of DNA strand breaks; however, none of the subsequent events of lymphocyte activation such as blast transformation and DNA synthesis occurred. These findings indicate that (1) cellular [NAD⁺] is a rate limiting factor in repair of DNA strand breaks in resting lymphocytes and (2) this repair is necessary but not sufficient for lymphocyte proliferation.     

Induction of 6-thioguanine resistance in synchronized human fibroblast cells treated with methyl methanesulfonate, N-acetoxy-2-acetylaminofluorene and N-ethyl-N′-nitro-N-nitrosoguanidine

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells progressed to the S phase after 16 h. DNA synthesis peaked 20 h after removal of nutrient stress and declined.Mutations were induced in S-phase cells by methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (NA-AAF) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Chemical treatments resulted in an increase in the absolute number of mutant colonies and in a dose-dependent mutation frequency. In this report, we show that NA-AAF evokes a temporal pattern of mutation in synchronized cells, with such mutations being induced only during the S phase. Evidence indicates that presence of S-phase cells in the treated cultures is a prerequisite for the induction of mutations.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9¹⁴-C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5–3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Enhancement by alkylating agents of chemical carcinogen transformation of hamster cells in culture

When Syrian hamster embryo cells were pretreated with a weak chemical carcinogen, methyl methanesulfonate (MMS) or ethyl methanesulfonate (EMS), or with a physical agent such as X-irradiation prior to being exposed to a potent cancer-producing chemical, transformation (crisscrossing of cells not seen in control) occurred up to nine times more often than when the cells were not pretreated. The degree of enhancement appears independent of carcinogen dose. The transformation frequency associated with the carcinogens benzo(a)pyrene (BP), dimethylbenz(a)anthracene (DMBA), 3-methylcholanthrene (MCA), N-acetoxy-2-acetylaminofluorene (AcAAF), and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was increased. There are similarities in the enhancement produced by pretreatment of hamster cells with X-irradiation and with alkylating agents: with both, maximum enhancement occurred approx. 48 h after treatment and lethality attributable to the pretreatment was 10–20% relative to control. However, enhancement produced by X-irradiation pretreatment was slightly greater than that obtained with MMS. The exact cause of the enhancement in transformation resulting from the interaction of these agents is not yet known, but the enhancement associated with MMS pretreatment cannot be related to partial cell synchronization or disruption in the cell cycle. Hamster cells pretreated with 250 μM of MMS demonstrated no alteration in normal cel DNA synthesis through 48-h post-treatment. Analysis of unscheduled DNA synthesis by autoradiography or by alkaline sucrose gradients indicated that the damaged DNA was rapidly repaired after treatment. Therefore, repair of DNA damage as it is now understood is probably not involved.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

13-cis-Retinoic Acid Affects Oxidation and DNA Damage in Oxidative-Positive SLE Lymphocytes But May Not Be Useful for Therapy

13-cis-Retinoic acid (13-CRA), a water-soluble vitamin A analog and 5′-lipoxygenase inhibitor, was tested in vitro for effects on excess oxidative metabolism and DNA damage in mitogen-stimulated lymphocytes from patients with systemic lupus erythematosus (SLE), because other 5′-lipoxygcnase enzyme inhibitors were shown to lower the excess oxidative metabolism in SLE cells. Excess chemiluminescence (CL) was abolished within minutes after the addition of 1 × 10⁻⁶ M 13-CRA in five of five CL-positive mitogen-stimulated SLE lymphocytes, and was lowered in five of eight samples after 48 to 72 h culture, Similarly, low concentrations of 13-CRA for 48-72 h largely prevented the S1 nuclease-sensitive DNA changes/DNA damage observed in CL-positive lupus lymphocytes in vitro. However, 13-CRA did not affect DNA damage in four of four CL-negative lymphocyte samples. 13-CRA, like other retinoic acid compounds, was known to stimulate B-cell activities in vivo and in vitro but effects on dividing lupus T cells had not been studied. 13-CRA further inhibited the diminished PHA-stimulated lupus T-cell growth in tissue culture at a concentration of 9 × 10⁻⁶ M in three of five lupus lymphocyte samples. 13-CRA has positive and negative effects on multiple aspects of the Immune system and it is not clear whether 13-CRA will have positive or adverse clinical effects on SLE patients. Close attention to vitamin A and vitamin "supplements" in patients with SLE may answer this question.