Alkylation of DNA leading to decreased sperm count

Event

Direct Essentiality Evidence

Indirect Essentiality Evidence

Uncertainties or Inconsistency

DNA alkylation (MIE)

Limited direct evidence

Exposure to alkylating agents leads to dose-dependent loss of germ cells and subsequent reductions in sperm counts (AO) across species; recovery may occur following removal of the stressor In rodents, treatment with alkylating agents at increasing doses results in dose-dependent decreases in testicular or epididymal sperm counts (Meistrich, 1982a, 1982b; Bucci and Meistrich, 1987). In rhesus macaques, similar progressive, dose-dependent declines have been observed following busulfan exposure, with higher doses leading to more persistent reductions in sperm production (Hermann et al., 2009). In cancer patients, the use of DNA alkylating drugs is strongly associated with lower sperm counts; such link is not observed in patients receiving non-alkylating drugs (Beaud et al., 2019).

Repeated exposure to an alkylating agent caused a marked reduction in sperm counts in mice, with gradual recovery following cessation of exposure, demonstrating reversibility of the AO after removal of the stressor (Yin et al., 2014).

Formation of alkyl DNA adducts in germ cells has been demonstrated in vivo; however, there are limited integrated measurements of the MIE, downstream KEs, the AO in the same studies. Consequently, progression through the pathway is often inferred from the known mechanisms of alkylating agents.

Inadequate repair (KE1)

Depletion of O6-alkylguanine-DNA alkyltransferase (AGT/MGMT) leads to corresponding alterations in DNA strand break (KE2) formation

Key studies: Roos et al. (2004) linked DNA alkylation (MIE), impaired repair (KE1), DNA strand breaks (KE2), and apoptosis (KE3) in proliferating human lymphocytes. Inactivation of MGMT increases persistence of alkylation-induced lesions, resulting in replication-dependent strand break formation and subsequent apoptosis.

Carlsson et al. (2025) showed that pharmacological inhibition of MGMT enhanced N-nitrosodimethylamine-induced formation of DNA adducts (MIE), DNA strand breaks (KE2), and micronucleus formation in HepG2-CYP2E1 human liver cells.

Modulation of DNA repair (KE1) or DDR pathways leads to concurrent increases in DNA strand breaks (KE2) and apoptosis (KE3)  

Knockdown or knockout of CDKN2AIP, a regulator of DNA repair, leads to increased double strand breaks (DSBs) and apoptosis in mouse Sertoli cells and male germ cells (Cao et al., 2022).

Greater impairment of DDR pathways (Nbn/Atm double deletion) results in more DSBs and apoptotic cells than Nbn single deletion in mouse neuronal tissues (Rodrigues et al., 2013). Similarly, single or double deletion of Apc/p53 (Méniel et al., 2015), bidirectional genetic modulation of CIRKIL/Ku70 (Xiao et al., 2023), inhibition of the PI3K/mTOR pathway (Liu et al., 2014), or homologous recombination (Stringer et al., 2020), result in more DSBs and apoptosis than wildtype or single deletion models.

DNA repair capacity is not measured directly in many studies. Accumulation of DSBs following impairment of DDR pathways is interpreted as evidence of insufficient repair.

DNA strand breaks (KE2)

Blocking signaling downstream of DNA strand breaks (KE2) prevents apoptosis (KE3) ATM inactivation prevents apoptosis in eye retina, despite the presence of DSBs (Rodrigues et al., 2013).

Modulation of the magnitude of DNA strand breaks (KE2) is associated with a corresponding change in apoptosis (KE3) The intervention studies listed in indirect evidence for KE1 also demonstrate a graded response-response relationship between DSBs and apoptosis.

TUNEL staining may detect both primary strand breaks and apoptotic DNA fragmentation, when KE2 and KE3 are measured at overlapping timepoints. More specific DSB markers (e.g., γH2AX) were used in several studies.

The absence of detectable DNA strand breaks may reflect limitations in the sampling time and assay specificity/sensitivity (e.g., the alkaline comet assay may miss DSBs, or the 24-hour sampling window may miss transient, repaired lesions).

Apoptosis (KE3)

Limited direct evidence

Attenuation of apoptosis (KE3) leads to recovery of sperm counts (AO) Key study (Oyovwi et al., 2023): Pharmacological attenuation of apoptosis using quercetin fully reversed testicular damage and restored sperm counts following levetiracetam exposure in rats. Multiple KEs were measured in this study, including sperm DNA fragmentation index (KE2; inferred from aniline blue staining), apoptotic markers (KE3: caspase-3, p53, cytochrome c, Bcl-2), and the AO (testicular sperm counts and histological evidence of germ cell loss). The concordant reversal of these endpoints following the intervention provides strong support for the progression across KEs. Additional intervention studies using antioxidants or protective agents (e.g., L-carnitine, plant extracts, quercetin, selenium nanoparticles, and probiotics) demonstrate that reducing apoptotic signaling is associated with improved sperm counts following exposure to chemotherapeutic agents, toxicants, or radiation (Yaman et al., 2018; Udefa et al., 2020; Ehghaghi et al., 2022).

Protective agents often suppress inflammation and oxidative stress simultaneously. It is unclear if sperm recovery is due to reduced apoptosis or these co-activated pathways, or both. Apoptotic markers are often measured in whole testis homogenates and the AO is likely caused by apoptosis of mixed testicular cell populations. Inappropriate sampling time and high variability in sperm count data may lead to "false negatives" (Gur et al., 2023).

Biological plausibility of KERs

Defining question

High (Strong)

Moderate

Low (Weak)

Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance.

KER is plausible based on analogy to accepted biological relationships, but scientific understanding is incomplete.

Empirical support for association between KEs, but the structural or functional relationship between them is not understood.

MIE  KE1: Alkylation, DNA leads to inadequate DNA repair

STRONG

Extensive evidence indicates that sufficiently high level of DNA alkylation can overwhelm cellular DNA repair machinery, leading to the persistence of DNA adducts and other unrepaired lesions. AGT, also known as MGMT in mammals, is an established suicide enzyme that can become saturated at high doses or after repeated exposure to alkylating agents, leading to inadequate DNA repair and accumulation of DNA alkyl adducts. This relationship is broadly conserved across species and cell types.

KE1  KE2: Inadequate DNA repair leads to Increase, DNA strand breaks

STRONG

DNA adducts and repair intermediates can accumulate when alkylation damage exceeds repair capacity in cells, including the depletion of AGT/MGMT. It is well established that persistence of unrepaired alkyl DNA lesions can interfere with DNA replication and promote replication fork stalling, leading to DNA strand breaks, which then activate DDR pathways. Extensive mechanistic evidence supports a causal relationship between inadequate DNA repair of alkylation-induced damage and increased DNA strand breaks.

KE2  KE3: Increase, DNA strand breaks leads to Apoptosis

STRONG

There is extensive mechanistic understanding of the DNA damage response pathways that link DNA strand breaks and apoptosis through both p53-dependent and independent mechanisms.

KE3  AO: Apoptosis leads to Decrease, Sperm Count

STRONG

Loss of testicular cells (e.g., developing germ cells and supportive somatic cells) through apoptosis disrupts normal testicular function to support spermatogenesis, resulting in a subsequent decrease in mature sperm output. These mechanisms are well established across mammalian systems.

MIE  AO: Alkylation, DNA leads to Decrease, Sperm Count

STRONG

The mechanistic linkage is conserved across species and supported by extensive knowledge of germ cell biology and toxicology. While alkylation damage can occur across all stages of spermatogenesis, effects on sperm counts are primarily driven by damage to proliferating and meiotic germ cells, whereas damage to post-meiotic cells predominantly affects sperm quality rather than quantity.

Essentiality of KEs

Defining question

High (Strong)

Moderate

Low (Weak)

Are downstream KEs and/or the AO prevented if an upstream KE is blocked?

Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs.

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE.

No or contradictory experimental evidence of the essentiality of any of the KEs.

AOP-level

MODERATE

Evidence supporting the essentiality of KEs is available from genetic and mechanistic studies. Modulation of DNA damage response and apoptotic pathways induce corresponding changes in downstream outcomes, including apoptosis and sperm counts. A limited number of studies provide more direct evidence of essentiality for specific KERs, while identifying a few essential signaling mediators involved in the transduction of DNA damage into apoptosis. However, such direct evidence is not consistently available across all KEs in the pathway, and much of the support remains indirect or context-specific.

Empirical support for KERs

Defining question

High (Strong)

Moderate

Low (Weak)

Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup> than that for KEdown? Inconsistencies?

Multiple studies showing dependent change in both events following exposure to a wide range of specific stressors. No or few critical data gaps or conflicting data.

Demonstrated dependent change in both events following exposure to a small number of stressors. Some inconsistencies with expected pattern that can be explained by various factors.

Limited or no studies reporting dependent change in both events following exposure to a specific stressor; and/or significant inconsistencies in empirical support across taxa and species that don’t align with hypothesized AOP.

MIE  KE1: Alkylation, DNA leads to inadequate DNA repair

STRONG

Inadequate DNA repair is measured indirectly through persistence of DNA adducts or increases in mutations that result from unrepaired DNA damage. Extensive evidence from somatic and germ cells supports this KER (in particular for temporal concordance), although quantitative dose-response concordance is less well characterized. There are no apparent inconsistencies. Numerous studies demonstrate that alkyl DNA adducts persist when repair capacity is exceeded or repair pathways are impaired. In particular, saturation or depletion of AGT/MGMT results in increased persistence of O6-alkylguanine adducts, providing strong support for this KER.

KE1  KE2: Inadequate DNA repair leads to Increase, DNA strand breaks

MODERATE

Limited in vivo data are available. However, multiple in vitro and genetic studies demonstrate that impairment of DNA repair or DDR pathways result in increased accumulation and persistence of DNA strand breaks in both somatic cells and germ cells following exposure to genotoxic stressors. In the context of DNA alkylation, saturation or depletion of AGT/MGMT leads to persistence of O6-alkylguanine lesions, which are subsequently converted into DNA strand breaks, consistent with temporal concordance. Studies involving AGT depletion and repair-deficient systems provide empirical support for the essential role of inadequate repair of alkylation DNA damage in the accumulation of DNA strand breaks.

KE2  KE3: Increase, DNA strand breaks leads to Apoptosis

STRONG

Temporal concordance is consistently observed, with DNA strand breaks occurring earlier or concurrently with apoptotic responses across in vitro somatic and germ cells, and rodent models. Dose concordance is supported, although dose-response data are limited in some studies. Evidence for incidence concordance is supported by a small number of studies, while others are limited by lack of appropriate measurements.

KE3  AO: Apoptosis leads to Reduce, Sperm Count

STRONG

Concordant changes between increased apoptosis and decreased sperm counts are have been consistently observed across multiple in vivo rodent studies. Temporal alignment is biologically supported, although it is often inferred rather than directly measured. Evidence for dose concordance is limited as many studies used a single exposure dose, preventing assessment of dose-dependent changes. Incidence concordance is generally not assessed, as both apoptosis and sperm count are typically reported as continuous outcomes (e.g., group means) rather than as the proportion of individual animals meeting predefined criteria for increased apoptosis or reduced sperm counts. Nevertheless, the consistency of the empirical evidence, together with multiple intervention studies demonstrating recovery of sperm counts following attenuation of apoptotic signaling, provides strong support for this KER.

MIE  AO: Alkylation, DNA leads to Decrease, Sperm Count

STRONG

Empirical evidence from both experimental animal models and human studies supports a consistent relationship between DNA alkylation and reduced sperm counts. Although direct measurement of both KEs in the same study is limited, extrapolation across studies involving exposure to well-characterized alkylating agents provides strong empirical support for temporal and dose concordance. Reduction in sperm counts occur after delays consistent with spermatogenic progression, and higher exposures lead to greater and more sustained decreases in sperm counts across multiple studies and stressors.

Modulating Factor

Influence on Outcome

KER(s) involved

DNA repair capacity

Determines the threshold at which DNA alkylation damage exceeds repair capacity and leads to insufficient or incorrect repair; lower capacity increases persistence and numbers of DNA lesions and the severity of downstream effects.

MIEKE1

KE1KE2

KE2KE3

Exposure dose/duration

Exposure doses and duration are strong modulating factors of the magnitude of effects across KEs. Higher doses or prolonged exposure are expected to increase cumulative DNA damage, which can lead to more severe downstream effects. At overtly cytotoxic concentrations (i.e., >50%, in the context of in vitro genotoxicity testing), non-specific cytotoxicity can occur, making it difficult to attribute apoptotic responses and the AO specifically to DNA alkylation.

All KERs

Stress-related pathways

Co-activation of parallel stress response pathways, oxidative stress, and/or inflammation, can amplify DNA damage and apoptosis, enhancing downstream effects independent of DNA alkylation.

KE1KE2

KE2KE3

Germ cell developmental stage

Proliferating or differentiating germ cells are more susceptible to DNA alkylation damage. Damage in spermatogonial stem cells (SSCs) leads to long-term or permanent reductions in sperm counts; damage in later stages results in transient effects or reduces sperm quality rather than quantity.

KE2KE3

KE3AO

MIEAO

Spermatogenic kinetics

The delay between DNA damage and observable reduction in sperm counts, as well as the time required for recovery (if SSCs survive), is determined by species-specific spermatogenic kinetics.

KE3AO

MIEAO