Association of SOD1 gene variants (50 bp Ins/Del, rs4817415, rs2070424, rs1041740, rs17880135) with plasma protein levels in vitiligo patients and their analysis in silico.

OBJECTIVE: Vitiligo is a common systemic, idiopathic autoimmune disease. The aim of this study was to analyze the frequency of variants of the superoxide dismutase 1 (SOD1) gene (50 bp Ins/Del, rs4817415, rs2070424, rs1041740, rs17880135) and circulating plasma protein levels through in-silico analysis. PATIENTS AND METHODS: Blood samples were collected from adult patients of both sexes with a clinical diagnosis of vitiligo. ELISA tests for SOD and analysis of gene variants by qPCR were compared to a disease-free reference group. RESULTS: The population analyzed was young people between 29 and 37 years old, with a higher percentage of women. The population was found in the Hardy-Weinberg equilibrium (HWE). The 50 bp Ins/Del, rs4817415, and rs2070424 variants showed no significant difference between groups (p > 0.05). Although, in the dominant model, the CT and CTTT genotypes of the rs1041740 and rs17880135 variants showed an association with susceptibility to vitiligo compared to the control. Plasma SOD levels showed significant differences between the groups, and when stratified according to the genotypes of each variant, there was a significant difference, except with the rs17880135 variant. The haplotypes InsCGTC and InsAGCC are shown to be risk factors for susceptibility to vitiligo. The in-silico analysis demonstrated that the rs4817415, rs2070424, rs1041740, and rs17880135 variants of the SOD1 gene participate in the modification of selected regulatory elements for differentiating the protein, transcription factors, and long non-coding RNA. CONCLUSIONS: Information regarding the pathogenesis of vitiligo helps recognize risk factors and identify the relationship of diagnostic markers of cell damage inherent to the disease. This will help improve aspects of prevention and the choice of treatment alternatives appropriate to each case.

Variants rs2758346, rs5746094, and rs2758331 of SOD2 gene: association study with breast cancer in a Mexican population and their analysis in silico.

OBJECTIVE: The aim of the study was to analyze the association between the superoxide dismutase 2 (SOD2) gene variants rs2758346, rs5746094, and rs2758331 and breast cancer (BC) in the Mexican population as well as to perform in silico assessments of the variants' potential impact. PATIENTS AND METHODS: We performed in silico analysis and analyzed 489 healthy women and 467 BC patients using TaqMan assays and Real-Time PCR. RESULTS: The TT genotype, the T allele of the rs2758346 variant, and the CC genotype of both rs5746094 and rs2758331 were identified as BC risk factors (p < 0.05). The TT and CTTT genotype of the rs2758346 variant stratified by the presence of ki-67 (> 20%), TCCC, and estrogen receptor (ER)-positive of the rs5746094 variant, and the CC and CT genotypes of rs2758331 stratified by menopause status and non-chemotherapy response were risk factors. The TTC and TTA haplotypes are risk factors for BC. In silico analysis revealed that the rs2758346, rs5746094, and rs2758331 variants could influence SOD2 gene regulation by transcription factors and circulating RNAs (circRNAs). CONCLUSIONS: The rs2758346, rs5746094, and rs2758331 variants of the SOD2 gene were associated with BC risk and could influence SOD2 regulation by transcription factors and circRNAs.

SOD1 gene variants rs4817415, rs2070424, and rs1041740 and their association with breast cancer risk.

OBJECTIVE: The aim of this investigation was to determine the frequency and association of the variants rs4817415, rs2070424, and rs1041740 of the SOD1 gene in healthy women and breast cancer (BC) patients. PATIENTS AND METHODS: Genomic DNA samples from 146 healthy women and 130 patients with BC were analyzed. RESULTS: GG genotype (OR 2.54, 95% CI 1.31-4.91, p = 0.0073) and the G allele (OR 1.37, 95% CI 1.09-1.73, p = 0.007) of the rs2070424 variant and CC genotype (OR 1.67, 95% CI 1.04-0.2.70, p = 0.0444) and allele C (OR 1.58, 95% CI 1.09-2.29, p = 0.0183) of the rs1041740 variant of SOD1 gene were associated as risk factors for BC susceptibility relative to the control group. Study groups comparison of the stratification by menopausal status showed an association of susceptibility to BC risk with carriers of the GG genotype (OR 2.9, 95% CI 1.11-7.81, p = 0.042) of the rs2070424 variant and with the premenopausal status of the study group and the TT (OR 2.89, 95% CI 1.73-4.85, p = 0.001) genotype of the rs1041740 variant. Furthermore, differences were observed in the patients with BC who were carriers of the CC genotype of the rs4817415 variant with elevated Ki-67 (≥ 20%) and who presented lymph node metastasis and stage III-IV BC (p<0.05). Two common haplotypes were identified in the study groups: CAC (protective factor), and CGC (risk factor) (p<0.05). CONCLUSIONS: The rs2070424 and rs1041740 variants of the SOD1 gene and the CGC haplotype were associated as risk susceptibility factors of BC in this sample analyzed.

Mutational spectrum of the iduronate-2-sulfatase gene in Mexican patients with Hunter syndrome.

OBJECTIVE: Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which is responsible for degrading heparan and dermatan sulfate. The IDS gene is located on chromosome Xq28; pathological variants in this gene mostly consist of missense mutations and small and larger deletions, which produce different phenotypes. However, there is only one record in our population concerning the molecular mechanism of this disease; a genotype-phenotype description is not available. PATIENTS AND METHODS: There were included 24 unrelated male patients; clinical features were recorded at a database, fluorometric IDS enzyme activity testing was done for each individual, followed by Sanger sequencing to identify mutations. RESULTS: The mutational spectrum was found in 16 out of 24 Mexican patients with MPS II, and its range of phenotypes was described. The most frequent variants were of the missense type. The most affected exons were exon 3 (c.275T>G, c.284_287del, c.325T>C), exon 8 (c.1035G>C, c.550G>A), exon 9 (c.1403G>C, c.1229_1229del), and exon 7 (c.979A>C; this variant has not been previously reported). Exon 5 (c.438C>T, a non-pathogenic variant) was the least frequent. It was also found that the most severely affected patients were those with large deletions (2 out of 24) [rsaIDS: IDSP1 (P164)x0, FMR1, AFF2 (P164)x2] involving genes and pseudogenes. We found 2 patients with a synonymous mutation in exon 4. CONCLUSIONS: Our results confirmed reports in the literature, since the most frequent variants were reported in exons 3 and 8. However, this result varies from one previous report in our population, which mentions large deletions and rearrangements as the most frequent alterations, since complex rearrangements were not found. According to what has been previously found, the most severely affected patients are those in which a whole gene has been deleted.

Association between rs61764370, rs9266, and rs140080026 polymorphisms of the KRAS gene and breast cancer risk in a Mexican population.

OBJECTIVE: Polymorphisms of the KRAS gene have been shown to be associated with cancer. However, their association with breast cancer (BC) has been inconsistent. The purpose of this study was to determine the frequency with which the rs61764370, rs9266, and rs140080026 polymorphisms of the KRAS gene are associated with BC in patients of the Mexican population. PATIENTS AND METHODS: The rs61764370 A>C or T>G and rs140080026 A>G polymorphisms were determined by Polymerase Chain Reaction (PCR), and the rs9266 A>G polymorphism was determined by DNA sequencing of healthy Mexican subjects and BC patients. RESULTS: We observed that 78% of BC patients are overweight and/or obese, 57% have metastatic lymph nodes, 64% have luminal A/B cancer subtypes, and 61% have stage III-IV cancer. The rs61764370 polymorphism was associated with BC susceptibility when the BC patients and the control group were compared for the AC genotype (p = 0.020), AC vs. AA genotypes (heterozygous model: p = 0.016), AC/CC genotype (dominant model: p = 0.002), and the C allele (p = 0.007). The AC/CC genotype (p = 0.018; rs61764370) and AG/GG genotype (p = 0.005; rs9266) were associated with age in BC patients ≥50 years old. The AC/CC (rs61764370) and AG/GG (rs9266) genotypes were classified by molecular subtype, TNM stage, miscarriage, lymph node metastasis, ductal type, and Ki-67. These classifications were also associated with BC patients, indicating that these factors may significantly contribute to BC risk. The AAA (OR 0.65, 95% CI 0.43-0.98, p = 0.039) and CAA (OR 3.25, 95% CI 1.13-9.36, p = 0.021) haplotypes were also associated with BC susceptibility. In addition, 94 polymorphisms were identified on the 3'UTR of the KRAS gene GRCh 38/hg3 (25,209,490-25,209,122) in BC (n = 112) and control (n = 113) samples. However, 92 of these polymorphisms have only expressed the major allele (wild-type allele). CONCLUSIONS: The rs61764370 polymorphism in the KRAS gene was associated with BC susceptibility in the Mexican population. The dominant model of the rs61764370 and rs9266 polymorphisms (classified by molecular subtype, miscarriage, TNM stage, lymph node metastasis, and Ki-67) could significantly contribute to BC risk in patients ≥50 years. The CAA haplotype could significantly contribute to BC risk in the Mexican population analyzed.

The rs1008562, rs2234671, and rs3138060 polymorphisms of the CXCR1 gene are associated with breast cancer risk in a Mexican population.

OBJECTIVE: The rs1008562, rs2234671 and rs3138060 polymorphisms of the CXCR1 gene have been shown to be associated with many diseases, but in breast cancer (BC) their association has not been detected. The purpose of this study was to determine the frequency and association of the rs1008562, rs2234671 and rs3138060 polymorphisms of CXCR1 gene in BC patients in the Mexican population. PATIENTS AND METHODS: The CXCR1 polymorphisms were determined by Polymerase Chain Reaction (PCR) and real time-PCR in healthy Mexican subjects and BC patients. RESULTS: The prevalent patron in BC patients was observed, the majority were overweight and obesity (72%) with metastatic lymph nodes (48%), luminal A/B subtypes (63%), and advanced stages (60%). Triple negative breast cancer (TNBC) patients: they were younger (58%) than 43 years old, overweight (33%), obesity (42%), ductal type histological (98%), metastasis to lymph nodes (47%), advanced stages III-IV (61%) and metastasis (33%). The rs2234671 polymorphism was associated with BC susceptibility when BC patients and the control group were compared for the CC genotype (p=0.037), CG (heterozygous model: p=0.018), GC/CC (dominant model: p=0.004), and the C allele (p=0.001), as well as the GC/CC genotype with hormone replace therapy (HRT, p=0.016). The rs3138060 polymorphism was associated with BC susceptibility for CG/GG genotype (dominant model: p=0.032) and G allele (p=0.018). Although the association between the dominant model of rs1008562, rs2234671, rs3138060 polymorphisms and BC patients and control was evident for tobacco and alcohol consumption (p<0.05). The rs1008562, rs2234671, and rs3138060 polymorphisms of the CXCR1 gene classified by molecular subtype and stage were also associated with BC patients, indicating that these factors may significantly contribute to BC risk. The CCC (OR 1.75, 95% CI 1.03- 2.97, p=0.046), GGG (OR 3.73, 95% CI 1.61- 8.65, p=0.0018) haplotypes were also associated with BC susceptibility. CONCLUSIONS: Rs2234671 and rs3138060 polymorphisms in the CXCR1 gene were associated with BC susceptibility in the Mexican population. The dominant model of the rs1008562, rs2234671 and rs3138060 polymorphisms could significantly contribute to BC risk in tobacco and alcohol consumption, molecular subtype and stage. The rs1008562, rs2234671 and rs3138060 polymorphisms, and the haplotypes CCC and GGG could significantly contribute to BC risk in the Mexican population analyzed.

Buccal micronucleus cytome assay of populations under chronic heavy metal and other metal exposure along the Santiago River, Mexico.

The Santiago River is one of the most contaminated rivers in Mexico, with heavy metal levels above the allowed limits. Scientific evidence indicates that chronic heavy metal exposure leads to cytogenotoxic effects. The aims of this study were to evaluate the genotoxic and cytotoxic effects of such exposure in buccal mucosa cells by micronucleus (MN) assay and to identify other nuclear abnormalities (NAs), such as nuclear buds (NBUDs), binucleated cells (BNs), pyknotic nuclei (PNs), karyorrhexis (KX), karyolysis (KL), and abnormally condensed chromatin (CC). Assays were performed on samples from four populations located alongside the Santiago River that are under chronic exposure to heavy metals and other metals (HMMs), and the results were compared with those of a population without exposure to HMMs. The exposed group showed increased frequencies of NAs (KX, CC, and KL), which are associated with cytotoxic damage, and NBUDs, which are associated with genotoxic damage. Increased frequencies of NBUDs and CC were observed in subjects from El Salto/Juanacatlán, Ocotlán, and Paso de Guadalupe, and an increase in KX frequency was observed in subjects from El Salto/Juanacatlán. Significant differences in KL frequency were observed in subjects from La Barca, El Salto/Juanacatlán, Paso de Guadalupe, and Ocotlán. Predictors for increased development of MNs and NBUDs were high concentrations of Al, Zn, and Cu. In conclusion, chronic exposure to HMMs, especially Al, Cu, and Zn, in the studied population could be related to increased frequencies of NAs, such as NBUDs, KX, CC, and KL, in the buccal mucosa cells.

Nuclear abnormalities in buccal mucosa cells of patients with type I and II diabetes treated with folic acid.

Diabetes mellitus (DM) is characterized by high blood glucose. Excessive production of free radicals may cause oxidative damage to DNA and other molecules, leading to complications of the disease. It may be possible to delay or reduce such damage by administration of antioxidants such as folic acid (FA). The objective of this study was to determine the effect of FA on nuclear abnormalities (NAs) in the oral mucosa of patients with DM. NAs (micronucleated cells, binucleated cells, pyknotic nuclei, karyorrhexis, karyolysis, abnormally condensed chromatin, and nuclear buds) were analyzed in 2000 cells from 45 healthy individuals (control group) and 55 patients with controlled or uncontrolled type I or II DM; 35 patients in the latter group were treated with FA. Samples were taken from the FA group before and after treatment. An increased rate of NAs was found in patients with DM in comparison with that of the control group (P<0.001). FA supplementation in patients with DM reduced the frequency of NAs (20.4 ± 8.0 before treatment vs. 10.5 ± 5.2 after treatment; P<0.001). The type I and type II DM and controlled and uncontrolled DM subgroups were analyzed in terms of sex, age, and smoking habit. The significantly reduced frequencies of buccal mucosa cells with micronuclei, binucleation, pyknosis, karyorrhexis, karyorrhexis+abnormally condensed chromatin, karyolysis, and nuclear buds produced by FA supplementation in DM patients (P<0.02) are consistent with the idea that free radicals are responsible for the increased frequency of NAs in DM patients.

Increased micronuclei and nuclear abnormalities in buccal mucosa and oxidative damage in saliva from patients with chronic and aggressive periodontal diseases.

BACKGROUND AND OBJECTIVE: Periodontal disease is a chronic bacterial infection characterized by connective tissue breakdown and alveolar bone destruction because of inflammatory and immune response caused by periodontopathogens and long-term release of reactive oxygen species. A high number of reactive oxygen species result in periodontal tissue damage through multiple mechanisms such as lipid peroxidation, protein denaturation and DNA damage. The aim of this study was to evaluate DNA and oxidative damage in subjects with chronic or aggressive periodontitis and healthy controls. MATERIAL AND METHODS: Buccal mucosa cells and whole saliva were collected from 160 subjects, who were divided into three groups: subjects with chronic periodontitis (CP) (n = 58), subjects with aggressive periodontitis (AgP) (n = 42) and a control group (n = 60). DNA damage was determined by counting micronuclei (MN) and nuclear abnormalities (NAs) in exfoliated cells, including binucleated cells, cells with nuclear buds and karyolitic, karyorrhectic, condensed chromatin and pyknotic cells. The degree of oxidative stress was determined by quantifying 8-hydroxy-2'-deoxyguanosine (8-OHdG) in whole saliva. RESULTS: Subjects with CP or AgP presented significantly more ( p < 0.05) MN and NAs and higher levels of 8-OHdG ( p < 0.05) compared with the control group. CONCLUSION: Our results indicate that subjects with periodontitis (CP or AgP) exhibited an increase in the frequency of MN, NAs and 8-OHdG, which is directly related to DNA damage. In addition, a positive correlation exists between oxidative stress produced by periodontitis disease and MN.

Chromosome instability in a patient with recurrent abortions.

Chromosomal aberrations are one of the recognized possible etiologic genetic causes of recurrent spontaneous abortions. Increased chromosome instability without constitutional chromosome abnormalities is uncommon in these couples. In this work we present a non consanguineous healthy couple with recurrent abortions without constitutional chromosome aberrations in which spontaneous and induced chromosome aberrations were observed in the female. Chromosome analysis was performed in the presence of different chromosome damage inductors such as gamma radiation, Uv light, and mitomycin-C. Alterations observed only in the female were: spontaneous and induced tetraradial chromosomes and increased chromosomal damage induced only by gamma radiation. Oral mucosa micronuclei were moderately increased in the female. Chromosome instability associated to abortion is proposed.

Oral sildenafil citrate lacks genotoxicity and cytotoxicity in a primate model: Callithrix jacchus.

OBJECTIVE: To determine sildenafil citrate (SC) genotoxicity and cytotoxicity in the Callithrix jacchus. STUDY DESIGN: Fifteen organisms were assigned to one of three groups as follows: experimental (25 mg/kg of SC); negative control (glucose solution 5%); and positive control (3 mg/kg of cytocine arabinoside). Systemic hemodynamic changes were monitored in each animal before and after each treatment. A drop of blood was obtained before and after the treatment at 24-120 h. Smears were made and the frequency of micronucleated erythrocytes (MNE), micronucleated polychromatic erythrocytes (MNPCE) and polychromatic erythrocytes (PCE) was counted. RESULTS: No significant differences in MNE, MNPCE and PCE were found in the group that received sildenafil and negative control. A significant increase in genotoxicity and cytotoxicity was observed in the positive control group. No changes were observed in systemic hemodynamic changes. CONCLUSION: The macro-dose of SC lacks genotoxic, cytotoxic or systemic hemodynamic changes effects in this species.

Evaluation of cisplatin + 5-FU, carboplatin + 5-FU, and ifosfamide + epirubicine regimens using the micronuclei test and nuclear abnormalities in the buccal mucosa.

In the present work, the micronuclei (MN) test was performed in buccal mucosal samples from patients with cancer, with (pre- and post-treatment) and without genotoxic chemotherapy (GC), identified micronucleated cells (MNC) and nuclear abnormalities (binucleated cells (BN), pycnosis (PN), "broken-egg" (BE), condensed chromatin (CC), karyorrhexis (KR), and karyolysis (KL). The objective was to evaluate the genotoxicity of cisplatin + 5-Fluorouracil (5-FU), carboplatin (CBP) + 5-Fluorouracil, and ifosfamide (IFO) + epirubicine (EPI) regimens. The ifosfamide + epirubicine regimen described here produced a micronucleogenic effect, whereas the regimens using platinum compounds were cytotoxic for buccal mucosal cells, which probably explain the absence of increase of micronucleated cells in these samples compared with basal levels. In patients with cancer (with and without genotoxic chemotherapy), the numbers of MNC, PN, KR, total nuclear abnormalities and KL increased, together with a decrease in BN cells and CC. On the other hand, as consequence of the cytotoxicity of the drugs, the number of binucleated cells decreased and the number of karyolytic cells increased. These results could be used as a cytotoxicity marker in the future studies for different drugs.

Evaluation of cisplatin + 5-FU, carboplatin + 5-FU, and ifosfamide + epirubicine regimens using the micronuclei test and nuclear abnormalities in the buccal mucosa.

In the present work, the micronuclei (MN) test was performed in buccal mucosal samples from patients with cancer, with (pre- and post-treatment) and without genotoxic chemotherapy (GC), identified micronucleated cells (MNC) and nuclear abnormalities (binucleated cells (BN), pycnosis (PN), "broken-egg" (BE), condensed chromatin (CC), karyorrhexis (KR), and karyolysis (KL)). The objective was to evaluate the genotoxicity of cisplatin+5-Fluorouracil (5-FU), carboplatin (CBP)+5-Fluorouracil, and ifosfamide (IFO)+epirubicine (EPI) regimens. The ifosfamide+epirubicine regimen described here produced a micronucleogenic effect, whereas the regimens using platinum compounds were cytotoxic for buccal mucosal cells, which probably explains the absence of increase of micronucleated cells in these samples compared with basal levels. In patients with cancer (with and without genotoxic chemotherapy), the numbers of micronucleated cells, pycnosis and karyolysis increased, together with a decrease in binucleated cells and chromatin-condensed. On the other hand, as consequence of the cytotoxicity of the drugs, the number of binucleated cells decreased and the number of karyolytic cells increased. These results could be used as a cytotoxicity marker in future studies for different drugs.

GSTT1 gene deletion is associated with lung cancer in Mexican patients.

Glutathione S-transferase (GST) is a dimeric detoxifying isoenzyme, involved in the deactivation of carcinogens, several tobacco-derived carcinogens, and xenobiotics. It catalyzes the reduction of glutathione to its thioester; thus, deficiency in GST activity due to homozygous deletion of the GSTT1 gene (null genotype) may play a role in the induction of lung cancer by smoking. We studied the distribution of GSTT1 gene deletion in peripheral blood DNA samples from 178 healthy controls (41 nonsmokers, 63 passive smokers and 74 smokers) and 52 lung cancer patients. Comparisons between groups showed that there was an increased lung cancer risk for individuals with the GSTT1 null genotype. Cancer patients showed significant differences when compared with controls: nonsmokers, passive smokers, and smokers. Twenty-one percent of lung cancer patients carried the deletion versus 2% among nonsmokers not exposed to passive smoking, 6% among passive smokers, and 5% among smokers. Thus, there is a significant association between this genotype and the possibility to risk of developing lung cancer.

Differences in the number of micronucleated erythrocytes among young and adult animals including humans. Spontaneous micronuclei in 43 species.

In our previous report we speculated about the possibility that some species had high levels of spontaneous micronucleated erythrocytes (MNE) just in a juvenile stage, this is, that the MNE diminish as the reticuloendothelial system matures. Here we show this effect in species including rat, rabbit, pig, dog, cat, gray squirrel, lion, giraffe, white-tailed deer, opossum and even human. The number of spontaneous MNE that we found in 43 species is shown, and the proportions of polychromatic and normochromatic. This is our third report on spontaneous MNE in different species. We obtained 189 peripheral blood samples of mammals, birds and reptiles. From 12 species we obtained only one sample, and 16 were reported previously, but now the size of the sample has been increased. The species with the highest spontaneous MNE were the Vietnamese potbelly pig (with the highest MNE number), Bengal tiger, capuchin monkey, puma, ferret, owl, hedgehog, squirrel monkey, pig and white-tailed deer. These species could be used as monitors for genotoxic events.

Variation of micronucleated erythrocytes in peripheral blood of Sciurus aureogaster in relation to age: an increment of micronucleated polychromatic erythrocytes after the administration of colchicine.

In some species, in which the human is included, the influence of age in the variation in the number of micronucleated erythrocytes (MNE) is known. In the present work we show how the process of aging influences the number of spontaneous MNE in the gray squirrel (Sciurus aureogaster). Because of the difficulty of knowing the age of each animal, 69 animals were weighed at their arrival to the laboratory and at the start of sample taking, with the supposition that the heaviest animals were the oldest and those with the lightest weight were the youngest. The major number of MNE was found in the younger animals, whereas the adults displayed less MNE (P < 0.0001). A group of 11 animals were sampled every 15 days over a period of 6 months, and the number of MNE were found to decrease with an increment in the weight in conformity with the time elapsed. These results showed that in the gray squirrel, the number of spontaneous MNE in peripheral blood depend on age. An additional interesting datum about the increment of MNE after the administration of colchicine is shown.

Spontaneous micronuclei in peripheral blood erythrocytes from 54 animal species (mammals, reptiles and birds): part two.

The normal numbers of micronucleated erythrocytes (MNE) observed in peripheral blood samples differ among species. This depends on the effectiveness of the spleen (or the rest of the reticuloendothelial system) to withdraw them from circulation. In our previous report, we assessed the number of MNE in the peripheral blood of 35 mammalian species. Here we show the results observed in 54 species including mammals, reptiles and birds. We obtained 212 peripheral blood samples from different species. In 14 species, only one individual was studied. Slides were stained with acridine orange. The total number of MNE (normo and polychromatic) in 10,000 erythrocytes per animal are shown. The species that display the higher MNE were: ocelote, lynx, owl, gray squirrel, hedgehog, lion, orange fronted parakeet and common barn owl. For this reason, these species could be tested as monitors for genotoxic events. Another interesting observation was that in the gray squirrel, we found the highest values of MNE in the smaller (younger) animals when compared with the larger (older) of the same species.