Testing the cytotoxic activity of FQ's based on the determination of the IC50 (inhibitory concentration), as well as IC25 and IC10 values. For comparative purposes also the effect of other commonly used antibiotics - streptomycin and tetracycline - on cell cultures. For this purpose, endoderm cells, neurons, cardiomyocytes and osteogenic cells in a wide range of concentrations will be tested. The MTS test will be used to assess the cytotoxic activity.
Knowing what complications Fluoroquinolones cause in mitochondrial replication and changes in genomic and mitochondrial DNA (study 2017),
we decided to organize a study on a representative group of people injured after treatment with Fluoroquinolones, in order to search for future FQAD therapies using iPSC technology. To this end, we have established the following strategy:
1) Isolation of epithelial cells from urine samples from patients.
2) Culture and preparation of urinary epithelial cells for the reprogramming process.
3) Reprogramming of epithelial cells from urine into induced pluripotent stem cells (iPSC).
4) Stabilization of obtained iPSC colonies, identification of pluripotency markers (SOX2, Oct3 / 4, TRA-1-81, TRA-1-60).
5) Banking the obtained Ipsc lines.
6) Differentiation of the iPSC line to neuronal stem cells (NSC).
7) Stabilization of the obtained NSC lines, identification of markers (Nestyna, SOX2).
8) Banking of NSC lines obtained.
Fluoroquinolones are a group of antibiotics that are widely prescribed to treat a variety of bacterial infections. While the intended target is bacteria, the Fluoroquinolones are also known to interact with the human genomic system by conjugating with Deoxyribonucleic acid (DNA). Given the crucial role of DNA in human body function, any damage may have serious health consequences.
The study we organized was conducted with subjects suffering from Fluoroquinolone Associated Disability (“FQAD”):
Determine if conjugates form between Levofloxacin, Ciprofloxacin and DNA.
(A1). Study for Fluoroquinolone DNA adduct. Human blood samples [50+ participants/samples from suspected cases FQAD]
(A2). Study for Fluoroquinolone mtDNA adduct. Human blood samples [50+ participants/samples from suspected cases FQAD]
(A3). Study for Fluoroquinolone genomic DNA adduct. Human blood samples [50+ participants from suspected cases FQAD, plus samples/4 children who had not taken a Fluoroquinolone, but their mothers did before or during pregnancy]
(A4). Control group who had never taken a Fluoroquinolone
-description of Fluoroquinolone DNA adduct
- methodology and strategy of study
- standard spectrogram Fluoroquinolone only
- spectrogram of control blood sample
-spectrograms representing the in vitro study for FQ DNA adduct after exposure of control blood to Fluoroquinolones
- spectrograms of Fluoroquinolone adduct to genomic and mitochondrial DNA
- diagrams showing the mechanism of Fluoroquinolone adduct to DNA
- summary, test results and substantiation
In the link below, you will find a sample of the results on the Levofloxacin adduct to mitochondria and genomic DNA along with methods used during this study:
Analysis of Human Genomic and Mitochondrial DNA was made by High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS / MS) for presence of adducts
Based on test for genomic FQ DNA adduct when exposed to Levofloxacin (abstract from FQ DNA adduct test result)
Fluoroquinolones are a group of antibiotics that were widely prescribed to treat a variety of bacterial infections. While the intended target is bacteria, the Fluoroquinolines were also known to interact with human genomic system by conjugating with Deoxyribonucleic acid (DNA). There were also numerous aberrations caused at various body tissue levels. Given the crucial role of DNA in human body function, any damage to it has serious health consequences. The objective of the present analysis of the patient's blood is to see if any conjugates (Adducts) are formed between Levofloxacin and DNA. The data presented here is limited to one patient and is subject to confirmation by a more elaborate investigation.
2. MATERIALS & METHODS
Commercially available kits from Sigma-Aldrich Company (Kit No. NA2020) and from Quiagen Inc. (Kit No. 37612) were employed for isolation and purification of DNA, using the manufacturer's standard procedures.
3. EXPERIMENTAL STRATEGY
A. Create an Adduct Molecule out of a reaction between Control Blood and Levofloxacin, and assign a chemical structure to the Adduct.
B. Analyze the Patient Blood for the presence of the Adduct and / or Levofloxacin.
C. If the Adduct molecule did not appear in tact but appeared in a modified form, examine possible relationship to the precursors.
Reference Standard of Levofloxacin yielded it characteristic spectrum with its molecular ion of m / z 362, and major daughter ion of m / z 342/344 (343) (Figure 1). Analysis of Control Blood
(Untreated with Levofloxacin) provided the background ions (Figure 2). Control Blood treated with Levofloxacin showed a product of m / z 494 (Figure 3). When the ion of m / z 494 was analyzed for its daughter ions, an ion of m / z 343 was obtained (Figure 4). This ion of m / z 343 was a common ion between Levofloxacin and the Adduct, thereby establishing the relation between these 2 molecules. HPLC-MS analysis of Patient's Genomic DNA showed several ions that were related to Levofloxacin (Figure 5). Comparable data was also seen with Patient's Mitochondrial DNA (Figure 6). Also, similar results were obtained with the 2 samples after acidic hydrolysis (Figures 7 and 8).
FDA Label of Levofloxacin states that: "Levofloxacin is mainly bound to serum albumin in humans." "Levofloxacin undergoes limited metabolism in humans and is primarily excreted as unchanged drug in the urine. Following oral administration, approximately 87% of an administered dose was recovered as unchanged drug in urine within 48 hours, whereas less than 4% of the dose was recovered in feces in 72 hours. Less than 5% of an administered dose was recovered in the urine as the desmethyl and N-oxide metabolites, the only metabolites identified in humans. These metabolites have little relevant pharmacological activity. "
The above situation needs to be reconciled with the general belief that certain toxic compounds may be stored in the adipose tissue and released later. The non-target effects of Levofloxacin may, therefore, be either of one time occurrence (immediately after the dosage), or of a minor recurrence (from storage). Given the pharmacological potency of Levofloxacin, its total elimination from patient's tissues may be important in assessing persistent disease condition.
An Adduct Molecule could be formed between Levofloxacin and Control Blood. The Adduct was structurally characterized, using Mass Spectrometry (HPLC-MS / MS). Although the exact configuration of the Adduct was not obvious in the Patient's DNA, the ions lend support to its origin to be Levofloxacin and Guanine (a portion of DNA molecule).
A proposed pathway towards the formation of the Adduct molecule is shown in Figures 9 and 10.
In tests with Ciprofloxacin in vivo tests, the mechanism of adduct formation between genomic and mitochondrial DNA was identical to that of Levofloxacin. The same components of guanine and the antibiotic, Ciprofloxacin, and major daughter ion were found. The crowning achievement of the research were the in vivo tests which overlapped with in vitro tests. All mtDNA genomic DNA patients previously exposed to Ciprofloxacin and Levofloxacin developed identical adducts as in the in vitro study. In the case of the children, it is necessary to perform tests on a larger group because the adduct also occurred in their case, even though they had never been treated with these antibiotics, but their mothers did before or during pregnancy. Another important observation was that the adduct may be formed regardless of the dose, form of its intake, age, brand and gender.
We know that other laboratories in the world have performed individual tests on FQ DNA adduct in humans, in various laboratories conducted in 2010 and 2019, 2020, 2021. Their results are in line with the research organized by our foundation. One laboratory has found that some individuals maintain Fluoroquinolone in lymphocytes even after 9 months of exposure. This phenomenon of accumulation and DNA adducts by Fluoroquinolones and mitochondrial damage is perceived by us as a serious global problem that poses a serious threat to the next generation.
For more information on adducts see the bottom of this page
Your support and contributions will help fund our latest research study currently in process.