Proposed modifications and incorporation of plasma Epstein-Barr virus DNA improve the TNM staging system for Epstein-Barr virus-related nasopharyngeal carcinoma.

BACKGROUND: The prognosis of patients who have Epstein-Barr virus (EBV)-related nasopharyngeal carcinoma (NPC) in which the tumor tissues harbor EBV have a better prognosis than those without EBV-related NPC. Therefore, the eighth edition of the TNM staging system could be modified for EBV-related NPC by incorporating the measurement of plasma EBV DNA. METHODS: In total, 979 patients with NPC who received intensity-modulated radiotherapy (IMRT) were retrospectively reviewed. Recursive partitioning analysis was conducted based on tumor (T) classification, lymph node (N) classification, and EBV DNA measurement to derive objectively the proposed stage groupings. The validity of the proposed stage groupings was confirmed in a prospective cohort of 550 consecutive patients who also received with IMRT. RESULTS: The pretreatment plasma EBV DNA level was identified as a significant, negative prognostic factor for progression-free survival and overall survival in univariate analysis (all P < .001) and multivariate analysis (all P < .05). Recursive partitioning analysis of the primary cohort to incorporate EBV DNA generated the following proposed stage groupings: stage RI (T1N0), RIIA (T2-T3N0 or T1-T3N1, EBV DNA ≤2000 copies/mL), stage RIIB (T2-T3N0 or T1-T3N1, EBV DNA >2000 copies/mL; T1-T3N2, EBV DNA ≤2000 copies/mL), stage RIII (T1-T3N2, EBV DNA >2000 copies/mL; T4N0-N2), and stage RIVA (any T and N3). In the validation cohort, the 5-year progression-free survival rate was 100%, 87.9%, 76.7%, 68.7%, and 50.4% for proposed stage RI, RIIA, RIIB, RIII, and RIV NPC, respectively (P < .001). Compared with the eighth edition TNM stage groupings, the proposed stage groupings incorporating EBV DNA provided better hazard consistency, hazard discrimination, outcome prediction, and sample size balance. CONCLUSIONS: The proposed stage groupings have better prognostic performance than the eighth edition of the TNM staging system. EBV DNA titers should be included in the TNM staging system to assess patients who have EBV-related NPC.

The effectiveness of riboflavin photochemical-mediated virus inactivation and changes in protein retention in fresh-frozen plasma treated using a flow-based treatment device.

BACKGROUND: A flow-based treatment device using riboflavin and ultraviolet (UV) light was developed to inactivate viruses in fresh-frozen plasma (FFP). The objective of this study was to evaluate the in vitro effectiveness of virus inactivation and changes in protein quality in FFP treated with this device. STUDY DESIGN AND METHODS: FFP-contaminating viruses were treated with riboflavin and UV light using a one-pass linear flow device. The infectivity of viruses was measured using established biologic assays. Real-time polymerase chain reaction (PCR) was performed to detect damage to viral nucleotides after treatment. Treated plasma was analyzed using standard coagulation assays. RESULTS: FFP treated at the UV dose of 3.6 J/cm(2) (J) exhibited a mean reduction of virus titer of more than 4 logs. The effectiveness increased significantly at higher doses. Real-time PCR showed that the cycle threshold values for both complete inactivation and virus recultivation were higher than that of the untreated sample. At doses of 3.6, 5.4, and 7.2 J, the protein recovery rates were 60.2 ± 8.6, 46.6 ± 9.4, and 28.0 ± 1.0% for fibrinogen; 67.0 ± 3.1, 57.3 ± 8.0, and 49.2 ± 3.8% for Factor VIII; 93.6 ± 2.8, 89.6 ± 6.1, and 86.5 ± 5.3% for antithrombin-III; and 72.1 ± 5.6, 59.8 ± 14.2, and 49.2 ± 8.4% for Protein C, respectively. CONCLUSION: The effectiveness of virus inactivation was enhanced, but total activity of plasma factors was reduced, in a UV dose-dependent manner.

[Influence of Low-Frequency Electromagnetic Field on DNA Molecules in Water Solutions].

It is shown that the amplicons of hepatitis virus DNA (hepatitis B virus, hepatitis C virus) are capable of inducing radiation after an exposure to electromagnetic fields in the frequency range from 3 to 30 Hz and the field strength, 24-40 A/m, registered by means of a chemiluminescence method. The most effect of the electromagnetic field on water solutions of the amplicons of hepatitis virus DNA occurs at the frequency of 9 Hz, the change in the hydration shell of DNA amplicons is observed. It is suggested that the change in the hydration shell of DNA amplicons exposed to the low-frequency electromagnetic field leads to restoration of hydrogen bonding, stitchings formation and DNA repair as a whole.

Modeling the repair of DNA strand breaks caused by γ-radiation in a minichromosome.

The objective of the studies described here was the development of a mathematical model which would fit experimental data for the repair of single and double strand breaks induced in DNA in living cells by exposure to ionizing radiation, and which would allow to better understand the processes of DNA repair. DNA breaks are believed to play the major role in radiation-induced lethality and formation of chromosome deletions, and are therefore crucial to the response of cells to radiotherapy. In an initial model which we reported on the basis of data for the repair of Epstein-Barr minichromosomes in irradiated Raji cells, we assumed that DNA breaks are induced only at the moment of irradiation and are later removed by repair systems. This work gives a development of that mathematical model which fits the experimental results more precisely and suggests strongly that DNA breaks are generated not only by direct irradiation but also later, probably by systems engaged in repair of oxidative damage.

Direct evidence that HSV DNA damaged by ultraviolet (UV) irradiation can be repaired in a cell type-dependent manner.

Infection of permissive cells, in tissue culture, with herpes simplex virus (HSV) has been reported to induce host DNA damage repair responses that are necessary for efficient viral replication. However, direct repair of the damaged viral DNA has not, to our knowledge, been shown. In this report, we detect and determine the amount of damaged HSV-1 DNA, following introduction of experimentally damaged HSV genomes into tissue cultures of permissive Vero, NGF differentiated PC12 cells and primary rat neurons, using a method of detection introduced here. The results show that HSV-1 strain 17 DNA containing UV-induced DNA damage is efficiently repaired, in Vero, but not NGF differentiated PC12 cells. The primary rat neuronal cultures were capable of repairing the damaged viral DNA, but with much less efficiency than did the permissive Vero cells. Moreover, by conducting the experiments with either an inhibitor of the HSV polymerase (phosphonoacetic acid [PAA]) or with a replication defective DNA polymerase mutant virus, HP66, the results suggest that repair can occur in the absence of a functional viral polymerase, although polymerase function seems to enhance the efficiency of the repair, in a replication independent manner. The possible significance of varying cell type mediated repair of viral DNA to viral pathogenesis is discussed.

Use of UV-C radiation to disinfect non-critical patient care items: a laboratory assessment of the Nanoclave Cabinet.

BACKGROUND: The near-patient environment is often heavily contaminated, yet the decontamination of near-patient surfaces and equipment is often poor. The Nanoclave Cabinet produces large amounts of ultraviolet-C (UV-C) radiation (53 W/m2) and is designed to rapidly disinfect individual items of clinical equipment. Controlled laboratory studies were conducted to assess its ability to eradicate a range of potential pathogens including Clostridium difficile spores and Adenovirus from different types of surface. METHODS: Each test surface was inoculated with known levels of vegetative bacteria (10(6) cfu/cm(2)), C. difficile spores (10(2)-10(6) cfu/cm(2)) or Adenovirus (10(9) viral genomes), placed in the Nanoclave Cabinet and exposed for up to 6 minutes to the UV-C light source. Survival of bacterial contaminants was determined via conventional cultivation techniques. Degradation of viral DNA was determined via PCR. Results were compared to the number of colonies or level of DNA recovered from non-exposed control surfaces. Experiments were repeated to incorporate organic soils and to compare the efficacy of the Nanoclave Cabinet to that of antimicrobial wipes. RESULTS: After exposing 8 common non-critical patient care items to two 30-second UV-C irradiation cycles, bacterial numbers on 40 of 51 target sites were consistently reduced to below detectable levels (≥ 4.7 log10 reduction). Bacterial load was reduced but still persisted on other sites. Objects that proved difficult to disinfect using the Nanoclave Cabinet (e.g. blood pressure cuff) were also difficult to disinfect using antimicrobial wipes. The efficacy of the Nanoclave Cabinet was not affected by the presence of organic soils. Clostridium difficile spores were more resistant to UV-C irradiation than vegetative bacteria. However, two 60-second irradiation cycles were sufficient to reduce the number of surface-associated spores from 10(3) cfu/cm(2) to below detectable levels. A 3 log10 reduction in detectable Adenovirus DNA was achieved within 3 minutes; after 6 minutes, viral DNA was undetectable. CONCLUSION: The results of this study suggest that the Nanoclave Cabinet can provide rapid and effective disinfection of some patient-related equipment. However, laboratory studies do not necessarily replicate 'in-use' conditions and further tests are required to assess the usability, acceptability and relative performance of the Nanoclave Cabinet when used in situ.

Titanium dioxide-assisted photocatalytic induction of prophages to lytic cycle.

The investigations on the kinetics of photocatalytic inactivation of bacteriophages, lactic bacteria and lysogenic lactic bacteria have shown that the rate of bacterial inactivation is ca. 10 times less than the inactivation of bacteriophages. Titania-assisted photorelease of bacteriophages from lysogenic bacteria proves that photogenerated reactive oxygen species affect the deoxyribonucleic acid (DNA) of bacteria before their deactivation. On this basis a novel photocatalytic method of a prophage induction to the lytic cycle and detection of lysogenic bacteria is proposed.

UV disinfection of adenoviruses: molecular indications of DNA damage efficiency.

Adenovirus is a focus of the water treatment community because of its resistance to standard, monochromatic low-pressure (LP) UV irradiation. Recent research has shown that polychromatic, medium-pressure (MP) UV sources are more effective than LP UV for disinfection of adenovirus when viral inactivation is measured using cell culture infectivity assays; however, UV-induced DNA damage may be repaired during cell culture infectivity assays, and this confounds interpretation of these results. Objectives of this work were to study adenoviral response to both LP and MP UV using (i) standard cell culture infectivity assays and (ii) a PCR assay to directly assess damage to the adenoviral genome without introducing the virus into cell culture. LP and MP UV dose response curves were determined for (i) log inactivation of the virus in cell culture and (ii) UV-induced lesions per kilobase of viral DNA as measured by the PCR assay. Results show that LP and MP UV are equally effective at damaging the genome; MP UV is more effective at inactivating adenovirus in cell culture. This work suggests that the higher disinfection efficacy of MP UV cannot be attributed to a difference in DNA damage induction. These results enhance our understanding of the fundamental mechanisms of UV disinfection of viruses-especially double-stranded DNA viruses that infect humans--and improve the ability of the water treatment community to protect public health.

Cellular and adenovirus dl312 DNA metabolism in cycling or mitotic human cultures exposed to supralethal gamma radiation.

Cellular repair of DNA damage due to lethal gamma irradiation was studied to reveal differences between strains and cell cycle stages that are otherwise difficult to detect. Cycling and metaphase-blocked cultures of normal fibroblasts and carcinoma cells were compared for repair of gamma sites (gamma radiation-induced nicks, breaks, and alkalilabile sites in DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation and at postirradiation incubations of 20-180 min. Fibroblasts from normal human skin or lung repaired gamma sites efficiently when cycling but did not repair them when blocked at mitosis. Bladder (253J) or lung (A549) carcinoma cells, unlike normal fibroblasts, repaired gamma sites efficiently even when blocked at mitosis. HeLa cells degraded their DNA soon after exposure at all doses tested, regardless of mitotic arrest. Whether the above differences in DNA repair between cell cycle stages and between strains result from differences in chromatin structure (cis effects) or from differences in the nuclear enzymatic environment (trans effects) could be resolved by placing an inert, extrachromosomal DNA molecule in the cell nucleus. Specifically, cis effects should be confined to the host chromosomes and would not be detected in the inert probe whereas trans effects should be detected in host chromosomes and inert probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus deletion mutant dl312, which does not proliferate in the absence of E1A complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were repaired efficiently in all hosts, regardless of mitotic arrest. Failure of mitosis-arrested fibroblasts to repair gamma sites was therefore due to a cis effect of chromatin organization rather than to a trans effect such as repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic carcinoma cells remained accessible to repair enzymes and nucleases alike. By means of these new tools, we should get a better understanding of higher-order chromatin management in normal and cancer cells.

Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA.

Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6 eV corresponding to 200-400 nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.

Molecular analysis of enhanced replication of UV-damaged simian virus 40 DNA in UV-treated mammalian cells.

Irradiation of simian virus 40 (SV40)-infected cells with low fluences of UV light (20 to 60 J/m2, inducing one to three pyrimidine dimers per SV40 genome) causes a dramatic inhibition of viral DNA replication. However, treatment of cells with UV radiation (20 J/m2) before infection with SV40 virus enhances the replication of UV-damaged viral DNA. To investigate the mechanism of this enhancement of replication, we analyzed the kinetics of synthesis and interconversion of viral replicative intermediates synthesized after UV irradiation of SV40-infected cells that had been pretreated with UV radiation. This enhancement did not appear to be due to an expansion of the size of the pool of replicative intermediates after irradiation of pretreated infected cells; the kinetics of incorporation of labeled thymidine into replicative intermediates were very similar after irradiation of infected control and pretreated cells. The major products of replication of SV40 DNA after UV irradiation at the low UV fluences used here were form II molecules with single-stranded gaps (relaxed circular intermediates). There did not appear to be a change in the proportion of these molecules synthesized when cells were pretreated with UV radiation. Thus, it is unlikely that a substantial amount of DNA synthesis occurs past pyrimidine dimers without leaving gaps. This conclusion is supported by the observation that the proportion of newly synthesized SV40 form I molecules that contain pyrimidine dimers was not increased in pretreated cells. Pulse-chase experiments suggested that there is a more efficient conversion of replicative intermediates into form I molecules in pretreated cells. This could be due to more efficient gap filling in relaxed circular intermediate molecules or to the release of blocked replication forks. Alternatively, the enhanced replication observed here may be due to an increase in the excision repair capacity of the pretreated cells.

Accommodation of pyrimidine dimers during replication of UV-damaged simian virus 40 DNA.

UV irradiation of simian virus 40-infected cells at fluences between 20 and 60 J/m2, which yield one to three pyrimidine dimers per simian virus 40 genome, leads to a fluence-dependent progressive decrease in simian virus 40 DNA replication as assayed by incorporation of [3H]deoxyribosylthymine into viral DNA. We used a variety of biochemical and biophysical techniques to show that this decrease is due to a block in the progression of replicative-intermediate molecules to completed form I molecules, with a concomitant decrease in the entry of molecules into the replicating pool. Despite this UV-induced inhibition of replication, some pyrimidine dimer-containing molecules become fully replicated after UV irradiation. The fraction of completed molecules containing dimers goes up with time such that by 3 h after a UV fluence of 40 J/m2, more than 50% of completed molecules contain pyrimidine dimers. We postulate that the cellular replication machinery can accommodate limited amounts of UV-induced damage and that the progressive decrease in simian virus 40 DNA synthesis after UV irradiation is due to the accumulation in the replication pool of blocked molecules containing levels of damage greater than that which can be tolerated.

Pyrimidine dimers block simian virus 40 replication forks.

UV light produces lesions, predominantly pyrimidine dimers, which inhibit DNA replication in mammalian cells. The mechanism of inhibition is controversial: is synthesis of a daughter strand halted at a lesion while the replication fork moves on and reinitiates downstream, or is fork progression itself blocked for some time at the site of a lesion? We directly addressed this question by using electron microscopy to examine the distances of replication forks from the origin in unirradiated and UV-irradiated simian virus 40 chromosomes. If UV lesions block replication fork progression, the forks should be asymmetrically located in a large fraction of the irradiated molecules; if replication forks move rapidly past lesions, the forks should be symmetrically located. A large fraction of the simian virus 40 replication forks in irradiated molecules were asymmetrically located, demonstrating that UV lesions present at the frequency of pyrimidine dimers block replication forks. As a mechanism for this fork blockage, we propose that polymerization of the leading strand makes a significant contribution to the energetics of fork movement, so any lesion in the template for the leading strand which blocks polymerization should also block fork movement.

Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation.

N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA.

Spectrum of complex DNA damages depends on the incident radiation.

Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.

Evaluation, in vitro, of the radioprotection of DNA from gamma-rays by naphazoline.

Design of compounds that can protect efficiently against gamma-rays irradiation is a great challenge. An ionizing event can cause variety of DNA damage scenarios leading to mutagenesis, cell death. 2-(1-Naphthylmethyl)-2-imidazoline (naphazoline, NP) is a drug belonging to the vasoregulator class, which was shown to be a very interesting compound in radioprotection. In order to highlight the NP radioprotective activity, a comparison of its ability to protect DNA against either gamma-irradiation or radicals generated by Fenton's reaction was made. Results show that NP inhibits efficiently the generation of DNA single-strand breaks and that NP is a potent radioprotector and also an hydroxyl radical scavenger.

Exposure of phage T7 to simulated space environment: the effect of vacuum and UV-C radiation.

The experiment "Phage and Uracil Response" (PUR) will be accommodated in the EXPOSE facility of the International Space Station (ISS). Its objective is to examine and quantify the effect of specific space conditions on bacteriophage T7 and isolated T7 DNA thin films. In order to define the environmental and technical requirements of the EXPOSE, the samples were subjected to the Experiment Verification Test (EVT). During EVT the samples were exposed to selected space conditions: high vacuum (10(-4) to 10(-6) Pa) and UV-C radiation (254 nm) alone and in combination. Characteristic changes in the absorption spectrum, in the electrophoretic pattern of DNA/phage and the decrease of the amount of PCR products have been detected indicating the damage of isolated and intraphage T7 DNA. Intraphage DNA is more sensitive to simulated space parameters than isolated T7 DNA in thin layers as well. We obtained substantial evidence that DNA lesions accumulate throughout exposure, and the amount of damage depends on the thickness of the layers. According to our preliminary results, the damages by exposure to conditions of dehydration and UV irradiation are larger than the sum of vacuum alone, or radiation alone case, suggesting a synergistic action of space vacuum and UV radiation with DNA being the critical target.

DNA damaging capability of hematoporphyrin towards DNAs of various accessibilities.

In this work we wanted to verify that photoactivation of DNA-non-binding porphyrin derivative hematoporphyrin IX (Hp) is able to induce damages in DNAs of various accessibilities such as B-conformation and superhelical isolated DNA, nucleoprotein complex and intracellular DNAs. It was found that photodynamic reaction of Hp results significant changes in thermal stability of isolated T7 DNA and induces single strand breaks in supercoiled Bluescript plasmid isolated from Escherichia coli cells. As optical melting measurements revealed, the irradiation of photosensitized T7 nucleoprotein complex leads to a destabilization of the protein capsid. The photodynamic reaction affected both the protein structure and DNA-protein interaction, however, the parameters corresponding to the DNA denaturation are not influenced. The accumulation of Hp in HeLa cells was followed by laser scanning confocal microscopy. The picture received is typical for lipophilic dyes. When Hp loaded cells were irradiated, a reduction of viability could be observed in a concentration and a light dose dependent manner; 12microM porphyrin induced almost complete cell killing after 30min irradiation. After similar treatment, alkaline agarose gel electrophoresis of isolated nuclear DNA did not show the presence of single strand breaks. The alkaline comet assay also failed to demonstrate any DNA damage in HeLa cells. We also considered the possibility of the generation of damages in intracellular SV40 DNA. According to the electropherograms there was no difference between the patterns of DNAs from treated and control samples.

High efficiency detection of bi-stranded abasic clusters in gamma-irradiated DNA by putrescine.

Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of approximately 1.7 or approximately 2 for DNA that had been gamma-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.

Reduced capacity to repair irradiated adenovirus in fibroblasts from xeroderma pigmentosum heterozygotes.

Xeroderma pigmentosum (XP) is one of a number of autosomal recessive syndromes in humans characterized by a marked predisposition to cancer. Fibroblasts from these patients show a defect in DNA repair. The XP heterozygotes also show elevated skin cancer incidence, but reports concerning their DNA repair capacity are conflicting. In this study, the DNA repair capacity of four XP heterozygotes was examined using a sensitive host cell reactivation technique. Unirradiated and irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens in fibroblasts from XP heterozygotes, XP homozygotes, and normals. A reduced host cell reactivation (of viral structural antigen production) for both ultraviolet- and gamma-irradiated Ad 2 was detected in four XP heterozygotes representing three different complementation groups as well as their XP homozygous children. The doses necessary to reduce the survival of viral structural antigen production by irradiated AD 2 to 37% in the XP heterozygous strains were expressed as a percentage of that obtained in normal strains and ranged from 55 to 82% for ultraviolet-irradiated Ad 2 and 71 to 79% for gamma-irradiated Ad 2. These results add further support to a direct relationship between cancer proneness and DNA repair defects and show the merits of this host cell reactivation technique in identifying XP heterozygotes. Identification of XP heterozygotes is of considerable public health interest not only in genetic counseling but also in the prevention of cancer.