The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein ...non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper’s content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.
Reaction of crotonaldehyde or two molecules of acetaldehyde with DNA generates 3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido1,2-apurine-10(3
H)one (
2, Scheme 1), which occurs ...in (6
R, 8
R) and (6
S, 8
S) configurations (Fig. 1). These diastereomers were site-specifically incorporated into oligonucleotides, which were then inserted into a double-stranded DNA vector for genotoxicity studies. Modified DNA was introduced into human xeroderma pigmentosum A (XPA) cells to allow replication. Analysis of progeny plasmid revealed that these DNA adducts inhibit DNA synthesis to similar degrees. (6
S, 8
S)-
2 miscodes more frequently than (6
R, 8
R)-
2: 10% versus 5%. For both adducts, major miscoding events were G
→
T transversions, but G
→
A transitions were also observed at a comparable level for (6
R, 8
R)-
2. G
→
C transversions were the second most common events for (6
S, 8
S)-
2. Comparison of these results with those of other 1,
N
2-propanodeoxyguanosine (PdG) adducts, which were evaluated by the same system, indicates that (i) their synthesis inhibiting potencies are stronger than that of the unsubstituted analog, 3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido1,2-apurine-10(3
H)one (
1, Scheme 1), but weaker than that of 3-(2′-deoxyribos-1′-yl)-5,6,7,8-tetrahydro-6-hydroxypyrimido1,2-apurine-10(3
H)one (
3, Scheme 1); (ii) both isomers of
2 are more miscoding than
1; (iii) the miscoding potency of (6
S, 8
S)-
2 is comparable to those of
3 and a model PdG 4 lacking a hydroxyl and a methyl group (Fig. 1). Therefore, considering the fact that
2 are formed endogenously as well as exogenously, they may play a significant role in aging and cancer in humans.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rats. ...NNK and NNAL require metabolic activation to express their carcinogenicity. Cytochrome P450-catalyzed alpha-hydroxylation at the methyl position of NNK or NNAL generates reactive intermediates, which alkylate DNA to form pyridyloxobutyl (POB)-DNA adducts or pyridylhydroxybutyl (PHB)-DNA adducts. NNK is metabolized to NNAL in a reversible and stereoselective manner, and the tissue-specific retention of (S)-NNAL is believed to be important to the carcinogenicity of NNK. In the present study, we investigated the formation of POB- and PHB-DNA adducts in extrahepatic tissues of F344 rats treated chronically with NNK and (R)- and (S)-NNAL (10 ppm in the drinking water, 1-20 weeks). POB- and PHB-DNA adducts were quantified in nasal olfactory mucosa, nasal respiratory mucosa, oral mucosa, and pancreas of treated rats. Adduct formation in the nasal respiratory mucosa exceeded that in the other tissues. O(2)-4-(3-Pyridyl)-4-oxobut-1-ylthymidine (O(2)-POB-dThd) or O(2)-4-(3-pyridyl)-4-hydroxybut-1-ylthymidine (O(2)-PHB-dThd) was the major adduct, followed by 7-4-(3-pyridyl)-4-oxobut-1-ylguanine (7-POB-Gua) or 7-4-(3-pyridyl)-4-hydroxybut-1-ylguanine (7-PHB-Gua). There was a remarkable similarity in adduct formation between the NNK and the (S)-NNAL groups, both of which were distinctively different from that in the (R)-NNAL group. For example, in the nasal olfactory mucosa, POB-DNA adduct levels in the NNK and (S)-NNAL groups were not significantly different from each other, while (R)-NNAL treatment generated 6-33 times lower amounts of POB-DNA adducts than did NNK treatment. In contrast, (R)-NNAL treatment produced significantly higher levels of PHB-DNA adducts than did NNK or (S)-NNAL treatment. Similar trends were observed in the nasal respiratory mucosa, oral mucosa, and pancreas. These results suggest extensive retention of (S)-NNAL in various tissues of NNK-treated rats and support a mechanism in which the preferential metabolism of NNK to (S)-NNAL, followed by sequestration of (S)-NNAL in the target tissues and reoxidation to NNK, is important to NNK tumorigenesis.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 2) are both potent pulmonary carcinogens in rats. The metabolism of ...NNK to NNAL is stereoselective and reversible, with (S)-NNAL being the major enantiomer formed from NNK. In rats, (R)-NNAL undergoes facile glucuronidation and is rapidly excreted in urine, whereas (S)-NNAL is preferentially retained in tissues and converted to NNK. We hypothesized that the lung carcinogenicity of NNK in the rat is due in part to the preferential retention of (S)-NNAL in the lung, the reconversion to NNK, and then the metabolic activation of NNK to pyridyloxobutyl (POB)-DNA adducts. We tested this hypothesis by treating male F344 rats with 10 ppm of NNK, (R)-NNAL, or (S)-NNAL in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in liver and lung DNA were quantified by HPLC-ESI-MS/MS. At each time point, total adduct levels were higher in the lung than in the liver. O2-4-(3-pyridyl)-4-oxobut-1-ylthymidine (O2-POB-dThd, 13) was the major adduct detected. Total adduct levels in the rats treated with (S)-NNAL were 0.6-1.3 times as great as those in the NNK group in the lung and 0.7-1.4 times in the liver, and 6-14 times higher than those in the (R)-NNAL group in the lung and 11-17 times in the liver. These results suggest that (S)-NNAL is stereoselectively retained in tissues. This study demonstrates for the first time the accumulation and persistence of specific POB-DNA adducts in the rat lung and liver during chronic treatment with NNK, (R)-NNAL, and (S)-NNAL and supports the hypothesis that the preferential retention of (S)-NNAL in the lung, followed by reconversion to NNK and then the metabolic activation of NNK is critical for lung carcinogenesis by NNK and NNAL.
NNN (1) is an esophageal carcinogen in rats. 2'-Hydroxylation of NNN is believed to be the major bioactivation pathway for NNN tumorigenicity. (S)-NNN is preferentially metabolized by ...2'-hydroxylation in cultured rat esophagus, whereas there is no preference for 2'-hydroxylation versus 5'-hydroxylation in the metabolism of (R)-NNN. 2'-Hydroxylation of NNN generates the reactive intermediate 4-oxo-4-(3-pyridyl)butanediazohydroxide (8), resulting in the formation of pyridyloxobutyl (POB)-DNA adducts. On the basis of these observations, we hypothesized that (S)-NNN treatment would produce higher levels of POB-DNA adducts than that by (R)-NNN in the rat esophagus. We tested this hypothesis by treating male F344 rats with 10 ppm of (R)-NNN or (S)-NNN in drinking water. After 1, 2, 5, 10, 16, or 20 weeks of treatment, POB-DNA adducts in esophageal, liver, and lung DNA were quantified by HPLC-ESI-MS/MS. In the rat esophagus, (S)-NNN treatment generated levels of POB-DNA adducts 3-5 times higher than (R)-NNN treatment, which supports our hypothesis. 7-4-(3-Pyridyl)-4-oxobut-1-ylguanine (7-POB-Gua, 14) was the major adduct detected, followed by O2-4-(3-pyridyl)-4-oxobut-1-ylthymidine (O2-POB-dThd, 11) and O2-4-(3-pyridyl)-4-oxobut-1-ylcytosine (POB-Cyt, 15). O6-4-(3-Pyridyl)-4-oxobut-1-yl-2'-deoxyguanosine (O6-POB-dGuo, 10) was not detected. The total POB-DNA adduct levels in the esophagus were 3-11 times higher than those in the liver for (R)-NNN and 2-6 times higher than those for (S)-NNN. In contrast to the esophagus and liver, (R)-NNN treatment produced more POB-DNA adducts than (S)-NNN treatment in the rat lung, which suggested an important role for cytochrome P450 2A3 in NNN metabolism in the rat lung. In both the liver and lung, O2-POB-dThd was the predominant adduct and accumulated during the experiment. The results of this study demonstrate that individual POB-DNA adducts form and persist in the esophagi, livers, and lungs of rats chronically treated with NNN enantiomers and demonstrate that (S)-NNN produces higher levels of POB-DNA adducts in the esophagus than (R)-NNN, suggesting that (S)-NNN is more tumorigenic than (R)-NNN to the rat esophagus.
N'-Nitrosonornicotine (NNN) is one of the most important strong carcinogens in tobacco products and is believed to play a significant role in the induction of esophageal cancer in smokers and oral ...cavity cancer in snuff dippers. NNN is metabolically activated through cytochrome P450-catalyzed alpha-hydroxylation. 2'-Hydroxylation produces a reactive intermediate 4-(3-pyridyl)-4-oxobutanediazohydroxide (7), which alkylates DNA to form pyridyloxobutyl (POB)-DNA adducts. DNA pyridyloxobutylation from NNN treatment, as measured by released 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8), has been observed in vitro and in vivo. In the present study, we have used liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to analyze specific POB-DNA adducts in the nasal olfactory, nasal respiratory, and oral mucosa of F344 rats treated chronically with (R)-NNN or (S)-NNN in the drinking water (10 ppm, 1-20 weeks). Adduct levels in the nasal respiratory mucosa exceeded those in the nasal olfactory and oral mucosa. (R)-NNN treatment generated 2-4 times more adducts in the nasal olfactory and respiratory mucosa than did (S)-NNN at most time points. O(2)-4-(3-Pyridyl)-4-oxobut-1-ylthymidine (O(2)-POB-dThd, 11) predominated in the nasal olfactory and respiratory mucosa, followed by 7-4-(3-pyridyl)-4-oxobut-1-ylguanine (7-POB-Gua, 14). Levels of O(2)-4-(3-pyridyl)-4-oxobut-1-ylcytosine (O(2)-POB-Cyt, 13) and O(6)-4-(3-pyridyl)-4-oxobut-1-yl-2'-deoxyguanosine (O(6)-POB-dGuo, 12) were significantly lower. In the oral mucosa, the opposite stereoselectivity was observed, with (S)-NNN treatment producing 3-5 times more POB-DNA adducts than did (R)-NNN. O(2)-POB-dThd and 7-POB-dGuo were the two major adducts, and their levels were similar. Overall, POB-DNA adduct formation in the nasal olfactory and nasal respiratory mucosa was similar to that previously observed in the lung, whereas in the oral mucosa, the trend resembled that in the esophagus. These results indicate that different mechanisms are involved in NNN metabolism and tumorigenesis in rat nasal and oral tissues. NNN enters the nasal mucosa through the circulation, and tissue-specific metabolism is important, while in the oral mucosa, direct exposure and local activation both play significant roles. Our results also support the potential importance of NNN as an oral carcinogen in people who use smokeless tobacco products.
The repair of acetaldehyde/crotonaldehyde-induced guanine (N2)-guanine (N2) interstrand cross-links (ICLs), 3-(2-deoxyribos-1-yl)-5,6,7,8-(N2-deoxyguanosyl)-6(R or ...S)-methylpyrimido1,2-alphapurine-10(3H)-one, was studied using a shuttle plasmid bearing a site-specific ICL. Since the authentic ICLs can revert to monoadducts, a chemically stable model ICL, 1,3-bis(2'-deoxyguanos-N2-yl)butane derivative, was also employed to probe the ICL repair mechanism. Since the removal of ICL depends on the nucleotide excision repair (NER) mechanism in Escherichia coli, the plasmid bearing the model ICL failed to yield transformants in NER-deficient host cells, proving the stability of this ICL in cells. The authentic ICLs yielded transformants in the NER-deficient hosts; therefore, these transformants are produced by plasmid bearing spontaneously reverted monoadducts. In contrast, in NER-deficient human cells, the model ICL was removed by an NER-independent repair pathway, which is unique to higher eukaryotes. This repair did not associate with a transcriptional event, but with replication. The analysis of repaired molecules revealed that the authentic and model ICLs were repaired mostly (>94%) in an error-free manner in both hosts. The major mutations that were observed were G --> T transversions targeting the cross-linked dG located in the lagging strand template. These results support one of the current models for the mammalian NER-independent ICL repair mechanism, in which a DNA endonuclease(s) unhooks an ICL from the leading strand template at a stalled replication fork site by incising on both sides of the ICL and then translesion synthesis is conducted across the "half-excised" ICL attached to the lagging strand template to restore DNA synthesis.
Exposure to the tobacco-related nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine is carcinogenic to humans. Metabolic activation of NNK leads to the ...formation of DNA adducts, which play a critical role in NNK carcinogenesis. Adducts specific to NNK result from covalent linkage of a pyridyloxobutyl (POB-1-yl) group to DNA. Furthermore, some such adducts are unstable, releasing the degradation product 4-hydroxy-1-(3-pyridyl)-1-butanone (4-HPB). Previous qualitative reports from our laboratory have established the chemical structures of the major POB-1-yl-DNA adducts. In this study, we have quantitated the levels of each of these adducts in vitro, as well as their contribution to the biomarker of DNA pyridyloxobutylation, 4-HPB. Standards for the POB-DNA adducts O(6)-(POB-1-yl)dGuo, 7-(POB-1-yl)Gua, O(2)-(POB-1-yl)dThd, and O(2)-(POB-1-yl)Cyt were synthesized and used to determine standard responses by reverse phase HPLC-electrospray ionization-tandem mass spectrometry (ESI-MS/MS). DNA was incubated with varying amounts of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone in the presence of an esterase, conditions favorable to the formation of an active pyridyloxobutylating agent. After sequential enzymatic and neutral thermal hydrolysis, isolation, and purification, the pyridyloxobutylated mixture was analyzed by HPLC-ESI-MS/MS to quantify the relative level of each of these four adducts as well as the released 4-HPB. The most abundant product was 4-HPB, which accounted for two-thirds of the analyzed mixture. The highest adduct levels measured were those of bases that result from loss of deoxyribose upon neutral thermal hydrolysis. These adducts, 7-(POB-1-yl)Gua and O(2)-(POB-1-yl)Cyt, comprised an average of 23 and 6% of the analyzed mixture, respectively. O(2)-(POB-1-yl)dThd and the mutagenic adduct O(6)-(POB-1-yl)dGuo were detected at the lowest levels, 4 and 2%, respectively. The relative levels of adducts determined in this study provide further insight regarding the chemical reactivity of the activated form of NNK with respect to DNA bases. Furthermore, the analytical standards and mass spectrometric methods used lay the groundwork for establishing a representative array of pyridyloxobutylation adducts as biomarkers of tobacco exposure in further biochemical and in vivo studies.
Tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine, are considered to be human carcinogens. Both compounds are metabolized to pyridyloxobutylating ...intermediates that react with DNA to form adducts such as 7-4-(3-pyridyl)-4-oxobut-1-ylguanine, O(2)-4-(3-pyridyl)-4-oxobut-1-ylcytosine, O(2)-4-(3-pyridyl)-4-oxobut-1-yl-2'-deoxythymidine (O(2)-pobdT), O(6)-4-(3-pyridyl)-4-oxobut-1-yl-2'-deoxyguanosine (O(6)-pobdG), and 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing adducts. The role of specific DNA adducts in the overall genotoxic activity of the pyridyloxobutylation pathway is not known. One adduct, O(6)-pobdG, is mutagenic. To characterize the mutagenic and cytotoxic properties of pyridyloxobutyl DNA adducts, the impact of DNA repair pathways on the cytotoxic and mutagenic properties of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), was investigated in Chinese hamster ovary cell lines proficient or deficient in O(6)-alkylguanine DNA alkyltransferase (AGT), deficient in both AGT and base excision repair (BER), or deficient in both AGT and nucleotide excision repair (NER). The repair of the four pyridyloxobutyl DNA adducts was determined in the same cell lines via sensitive LC-MS/MS methods. NNKOAc was more cytotoxic in the cell lines lacking AGT, BER, and NER repair pathways. It also induced more mutations in the hprt gene in the BER- and NER-deficient cell lines. However, AGT expression did not influence NNKOAc's mutagenicity despite efficient repair of O(6)-pobdG. Analysis of the hprt mutational spectra indicated that NNKOAc primarily caused point mutations at AT base pairs. GC to AT transition mutations were a minor contributor to the overall mutation spectrum, providing a rationale for the observation that AGT does not protect against the overall mutagenic properties of NNKOAc in this model system. The only adduct affected by the absence of effective NER was O(2)-pobdT. Slower repair of O(2)-pobdT in NER-deficient cells was associated with increased AT to TA transversion mutations, supporting the hypothesis that these mutations are caused by O(2)-pobdT. Together, these data support a hypothesis that the pyridyloxobutylation pathway generates multiple mutagenic and toxic adducts.
Acetaldehyde (AA), occurring widely in the human environment, is a mutagen and carcinogen. AA can react with DNA to form AA-DNA adducts. Several types of adducts, including an interstrand cross-link ...3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)-6-methylpyrimido1,2-apurine-10(3H)one (7), have been previously characterized by our laboratory. We hypothesize that cross-link 7 may be involved in determining the mutagenic and carcinogenic properties of AA. To address this question, the double-stranded oligonucleotide 13, bearing cross-link 7, was synthesized in a sequence appropriate for mutagenicity studies in human cells. Oligonucleotide 9, containing 2-fluoro-O6-(trimethylsilylethyl)deoxyinosine (dIno), was reacted with 4-amino-1,2-pentanediol, followed by treatment with NaIO4. The resulting oligonucleotide 11 containing the 1,N2-propano-deoxyguanosine (dGuo) 5 was incubated with the complementary oligonucleotide 12 to give the desired cross-link 13, which was characterized by negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) and enzymatic hydrolysis to cross-link 7. The formation of cross-link 13 at 5'-CpG-3' was confirmed by incubation of 11 with 15N512 containing a 5'-Cp15N5G-3' sequence. The formation of cross-link 13 was reversible. Therefore, oligonucleotide 24 containing the irreversible analogue of cross-link 7, 1,3-bis(2'-deoxyguanos-N2-yl)butane, was synthesized for use as a control in the mutagenicity studies. Oligonucleotide 21 was reacted with 1,3-diaminobutane dihydrochloride, followed by incubation with the complementary oligonucleotide 23, to give 24. To determine the optimum distance and orientation for cross-link formation, six oligonucleotides, containing 5 at the i + 1, i + 2, and i + 3 or the i - 1, i - 2, and i - 3 positions relative to dGuo in the complementary strand, were 5'-end labeled with gamma-32PATP, followed by incubation of each labeled oligonulceotide with its complementary strand and then analysis by denaturing polyacrylamide gel electrophoresis. Only the oligonucleotide containing 5'-Cp5-3' formed the cross-link with the complementary 5'-CpG-3' sequence. The results of this study confirm the structure of an AA-derived DNA cross-link, supply characterized cross-link-containing oligonucleotides for mutagenicity studies, and provide information on the optimum distance and orientation for cross-link formation.
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