https://www.koreabiomed.com/news/articleView.html?idxno=21377
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Any knowledge of the limit that manufactures have been working against?
Prof @ymleefn any insights?
EMA CMDh requested to NcWP for N-nitroso tamsulosin AI limit derivation in its April 2023 meeting. So its AI is in progress work…
In Australia, the TGA has set a limit of 18ng per day
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Health Canada published a limit of 18ng per day.
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Additionally ANVISA also include Nitroso tamsulosine in its recently updated (Version 3) guidance on Control of Nitrosoamine impurities with same AI i.e 18 ng/day as like health Canada and TGA
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We have already published papers regarding the simultaneously development of tamsulosin / together with other potentially nitrosamine contaminated drugs -and the subsequent skin cancer development !
Here:
There are some errata in this paper. For example:
- Citation 18 (Li 2023) contains Ames test positive data on N-nitroso-propanolol, whereas you say this paper shows Ames test negativity on N-nitroso-propanolol:
- The statement that nitrosamines exist that can only be activated by irradiation is supported by NMOR as an example. However, for NMOR literature exists supporting enzyme activation, literature on NMOR mutagenicity/carcinogenicity studies can counter the relevance of this citation to support the statement:
I’m also missing reference to recent literature studying the correlation between nitrosamine carcinogenicity and Ames test data:
At the same time, it is difficult to understand the critique on the Ames test for nitrosamines, now that the embraced hypothesis of photocarcinogenicity is also often investigated with such or similar in vitro test systems using irradiation to activate the nitrosamine (this includes citation 31 on NMOR though non-cited literature exist to support the NMOR accumulates in skin)?
I’m also wondering why nitrosoproline literature is not considered?
Though the mechanism described for irradiation activated metabolization of nitrosoproline is different to the described irradiation-mediated alpha-hydroxylation of NMOR with a link with phosphate, it seems this case is well-described in peer reviewed literature.
Considering that proline can endogenously nitrosate to nitrosoproline (cf. also the use of nitrosoproline as an urinary biomarker) and that proline is obviously quite abundant (but nitrosoproline not per se accumulating in skin maybe), how likely is the contribution of NDSRIs really in absence of proof exposure exists and that they are accumulating in skin (or at least more than nitrosoproline) and can be photoactivated (at leas as good as activated structures, cf. for example COOH mechanistic role in proline)? Shouldn’t in absence of such data bigger populations be studied, studying the prevalence of skin cancer in groups taking and not taking the medicine, with a reflection on geography ?
- doi.org/10.1016/j.mrgentox.2017.06.0037
- Redirecting
- Redirecting
- Redirecting
- Mutation, DNA strand cleavage and nitric oxide formation caused by N -nitrosoproline with sunlight: a possible mechanism of UVA carcinogenicity | Carcinogenesis | Oxford Academic
Any particular reason to not consider the photosensitivity of tamsulosin itself?
Hi! Agree to some extend!
Commenting point 2 firstly!
Starting with nitrosomorpholone and Phototoxicity ! Activation of Phototreated NMOR was directly mutagenic without metabolic activation!
Metabolic activation is a of course also possible! And carcinogenicity after that.
I have to write it more accurate !
However and as it follows - that means that all nitrosamines and their DNA addicts has to be checked for photocarcinogenic activity : means prior metabolisation and after metabolisation ! That’s means that many of the nitrosamines and ndsris in drugs can probably have carcinogenic effect also prior the metabolisation in the liver!
This will be a huge work , I suppose…
Regarding point 1 : Ames test was negative for Nitroso proparanolol:
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Regul Toxicol Pharmacol
. 2023 Jun:141:105410.
doi: 10.1016/j.yrtph.2023.105410.Epub 2023 May 18.
Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and human in vitro assays
Xilin Li 1, Yuan Le 2, Ji-Eun Seo 2, Xiaoqing Guo 2, Yuxi Li 2, Si Chen 2, Roberta A Mittelstaedt 2, Nyosha Moore 2, Sharon Guerrero 2, Audrey Sims 2, Sruthi T King 3, Aisar H Atrakchi 3, Timothy J McGovern 3, Karen L Davis-Bruno 3, David A Keire 3, Rosalie K Elespuru 4, Robert H Heflich 2, Nan Mei 5
Affiliations expand
- PMID: 37210026
- DOI: 10.1016/j.yrtph.2023.105410
Full text linksCite
Abstract
Propranolol is a widely used β-blocker that can generate a nitrosated derivative, N-nitroso propranolol (NNP). NNP has been reported to be negative in the bacterial reverse mutation test (the Ames test) but genotoxic in other in vitro assays. In the current study, we systematically examined the in vitro mutagenicity and genotoxicity of NNP using several modifications of the Ames test known to affect the mutagenicity of nitrosamines, as well as a battery of genotoxicity tests using human cells. We found that NNP induced concentration-dependent mutations in the Ames test, both in two tester strains that detect base pair substitutions, TA1535 and TA100, as well as in the TA98 frameshift-detector strain. Although positive results were seen with rat liver S9, the hamster liver S9 fraction was more effective in bio-transforming NNP into a reactive mutagen. NNP also induced micronuclei and gene mutations in human lymphoblastoid TK6 cells in the presence of hamster liver S9. Using a panel of TK6 cell lines that each expresses a different human cytochrome P450 (CYP), CYP2C19 was identified as the most active enzyme in the bioactivation of NNP to a genotoxicant among those tested. NNP also induced concentration-dependent DNA strand breakage in metabolically competent 2-dimensional (2D) and 3D cultures of human HepaRG cells. This study indicates that NNP is genotoxic in a variety of bacterial and mammalian systems. Thus, NNP is a mutagenic and genotoxic nitrosamine and a potential human carcinogen.
Keywords: Chromosomal damage; Cytochrome P450s; Gene mutation; Hamster liver S9; N-Nitroso propranolol; Nitrosamine impurities.
In the abstract u can found the data that NN Propranolol has been already reported to have negative Ames test-
Please , read it carefully!
Thanks
Photocarcinogenisity has to be tested firstly before the activation of each nitrosamine or ndsri and after that the dna adducts also require a test !
Reason: we have both positive reactions for NMOR- before metabolisation and after photoactivation!
Photo activation of nitrosamines seems to be a completely new way for mutagenicity and carcinogenicity…
Hope that this is more than clear!
Greetings
Methylated purines or tumour tissue from patients taking nitrosamine contaminated drugs can be easily tested for methylating agents even in tumourous tissue as it has been done 45 years ago:
Methylated purines in human liver DNA after probable dimethylnitrosamine poisoning
- PMID: 7427930
Cite
Abstract
DNA, isolated from two samples of human liver obtained from a suspected dimethylnitrosamine poisoning, contained 1363 to 1373 micromol of 7-methylguanine per mol of guanine and 273 to 317 micromol of O6-methylguanine per mol of guanine. Liver and kidney DNA obtained from unrelated cases contained no detectable methylated purines. From the DNA methylation levels, it is estimated that the dimethylnitrosamine-poisoning victim had been exposed to a dose of 20 mg or more of dimethylnitrosamine per kg of body weight. The results indicate for the first time that humans, like rodents, appear to activate dimethylnitrosamine metabolically to a strong methylating agent, resulting in methylation of liver DNA at both the 7- and O6 positions of guanine.
[PubMed](Disclaimer - PubMed
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I can repeat:
- Citation 18 (Li 2023) contains Ames test positive data on N-nitroso-propanolol, whereas you say this paper shows Ames test negativity on N-nitroso-propanolol:
Please read the supplementary information of Li 2023.
You are referring to “completely new way”, yet citing only NMOR photo-induced genotoxicity (which has been indicated to be linked with alpha-hydroxylation and phosphate so far (contrary to nitrosoproline where the COOH plays a mechanistic role) - thus showing a mechanistic parallel and is not new science) and referring to DNA alkylation checks (linked to NDMA exposure) which can’t distinct with enzyme-based mechanisms and are in fact indicative of metabolic activation? Of note, NDSRIs are not per se theoretically methylating agents, this depends on the N-substitution patterns and the likelihood of chain-shortening mechanisms, but can be checked by building in a radiolabeled carbon as part of the alpha-CH2 (if present) and checking for radiolabeled methylated guanine (this again not having an automatic link with proving photo-activation in the standard design of experiments).
[quote=“ccdw, post:14, topic:6510”]
You are referring to “completely new way”, yet citing only NMOR photo-induced genotoxicity (which has been indicated to be linked with alpha-hydroxylation and phosphate so far (contrary to nitrosoproline where the COOH plays a mechanistic role) - thus showing a mechanistic parallel and is not new science) and referring to DNA alkylation checks (linked to NDMA exposure) which can’t distinct with enzyme-based mechanisms and are in fact indicative of metabolic activation? Of note, NDSRIs are not per se theoretically methylating agents, this depends on the N-substitution patterns and the likelihood of chain-shortening mechanisms, but can be checked by building in a radiolabeled carbon as part of the alpha-CH2 (if present) and checking for radiolabeled methylated guanine (this again not having an automatic link with proving photo-activation in the standard design …
In this paper the Phototoxicity has been not linked to metabolic activation!
The alpha hydroxylation isn’t presented - I have seen du the abstract to take a careful look!
It’s a completely new understanding that nitrosamines might be carcinogenic without metabolic activation!
That was the idea!
That’s why before activation in the liver - Phototoxicity has to be checked !
Additionally After the activation we have two important things to be probably checked
- Phototoxicity of the dna aducts
- availability of dna adducts/ methylated products for example in tumor tissue
Hope that’s more than clear!
Every paper I have seen on NMOR photo-induced genotoxicity that discusses mechanistic explanations on what happens with NMOR after photo-induction to explain mutagenicity, discusses alpha-hydroxylated NMOR as a metabolite (as proven experimentally) and the dependency on phosphate or similar ions (as proven experimentally) (conjugation link?). What paper presents something else that goes further than suggesting radical-based chemistry?
In proline there is anchimeric assistance of the COOH hence the significant difference in metabolisation mechanism upon photo-activation. Noteworthy, this literature is also much more developed.
Mechanistic understanding on what happens with a nitrosamine when you irradiate it, seems important to me to evaluate if this differentiates the risk or not, to be able to ask the question: could one of the enzymes typically present in liver homogenate (based on proteomics characterisation data) activate a pathway to the same type of metabolite?
Is alpha hydroxylation a result only of a metabolic activation?
Or not ?
Stay 
cool! Don’t be angry !
Do you have a pdf of the paper I attached / I send you the abstract already
I think that this paper indicates on the possible genotoxic action of NMOR without the metabolic activation:
Try to obtain the pdf - we have to check this!
I depends from that patients tumour tissue has to be investigate with all available methods in medical
Science in order to check for the methylated end products / dna adducts in the tissue !
All of them
Has to be checked also for Phototoxicity - it’s good to have ir on this indeed? The conversation has the purpose not only to criticise some not completely precise mentioned statements , but to give the opportunity to comment the new suggestions!
I miss this in ur comments- miss it completely
I assume you read this paper you cite in your published work? I can’t provide copies of papers subject to copy rights, hopefully you can finally get a copy, as it is such an important reflection of the NMOR state of the art for you.
I have already tried to suggest a few times that this new 2024 paper does build further on the finding that irradiation is a non-enzymatic way to alpha-hydroxy-NMOR, when conjugated with phosphate or acetate this gives a fairly stable direct acting mutagen (in vitro, absence of enzymes avoids that alpha-hydroxy O-conjugated NMOR gets decomposed fastly).
For the enzymatic pathway: NMOR is metabolized mainly via alpha- and
beta-hydroxylation (O-conjugation as always a possibility). The hydroxylation product of NMOR is unstable and decomposes quickly to react with biological molecules, including DNA, resulting in further decomposition products. But alpha-hydroxy NMOR is effectively a shared “metabolite” photo-route and enzyme-route. So basing risk assessment for medicines on alpha-hydroxy NMOR enzyme-route can still be justified and in reality for the medicine exposure route if applicable, enzymes will not be equally suppressed (whereas mechanisms for UV protection can also play).
It is not because the photoactivation pathway could replace (cf. suppression of enzymes in experiments needed to see the effect) in vivo the role of enzymes (as seen by the in vivo micronucleus test with enzyme suppression but photoactivation), that the “metabolites” causing the effect are different. This helps to explain why this 2024 paper stresses the importance of environmental risk assessment for nitrosamines, as in the environment NMOR can be activated to a direct acting mutagen (like premetabolised) under the influence of irradiation. It also explains why the authors are focused on the stability of irradiation activated mutagenicity of NMOR in absence of enzymatic activity. Irradation risk on the medicine or its precursors directly (transforming NMOR impurities when present (and allowing conjugation)) is probably uncommon and in vivo this mechanism is clearly in competition with the enzymatic routes.
The 2024 paper does not move away from the theories on alpha-hydroxy and conjugated NMOR (similar NPIP). (I do recognise irradation-based “metabolites” of nitrosoproline are significantly differently as described in literature, cf. anchimeric assistance of COOH, and that you might still be doing a deep dive in the vast amount of research on this topic. In fact the (even in vitro) differences NMOR vs. NPRO already show that this is not one-shoe-fits all and that NMOR can’t be easily extrapolated to many NDSRIs).
I believe data generation is as important as hypothesis building, but that also hypothesis building should depart from available data and is thus dependent on data interpretation, which is where we are lost in translation (next to a few other things, like DNA alkylation risks linked to nitrosamines that are evaluated is broader than methylation).
I think this will also helpful.
180424_ACT-2023_Poster440-1.pdf (1.4 MB)